KR20050008651A - Methods for detecting genome-wide sequence variations associated with a phenotype - Google Patents

Abstract

The present invention provides a method for determining genome-wide sequence variation in a hypothesis that is not related to the phenotype of the species. In the methods of the present invention, each restriction fragment set in a subpopulation of an individual with the phenotype is generated by digesting the nucleic acid of the individual using one or more different restriction enzymes. The set of restriction sequences tags of the individual is then determined from the set of restriction fragments. Restriction sequence tags of subpopulations of organisms are compared and classified into one or more groups comprising restriction sequence tags comprising homologous sequences. One or more groups of restriction sequence tags obtained identify sequence variations associated with the phenotype. For example, the methods of the present invention can be used to analyze large numbers of sequence variations in a sample of many patients to identify faint genetic risk factors.

Description

Methods for detecting genome-wide sequence variations associated with a phenotype

Molecular approaches for genetic analysis track nucleotide sequence variations that occur naturally and randomly in the genome of life. Knowledge of DNA polymorphism between individuals and groups is important in understanding the complex linkage between genotypes and phenotypes. Without complete data on sequence variation, we rely on our ability to recognize 'nearby' markers that make it possible to infer certain relevant positions or positions of irregular sequence variations. The informativeness of a marker depends on the size of linkage disequilibrium (LD). Markers can be used in lingage studies to study candidate genes and related studies to identify functional allelic variation of candidate genes that affect inter-individual variation.

In order to correlate the side effects of drug treatment and disease susceptibility with the genetic makeup of individuals, it is essential to monitor the differences in genomes between individuals. Current approaches include monitoring large sets of genetic markers, for example thousands of single nucleotide polymorphisms (SNPs) that are evenly distributed in the genome. These SNPs are monitored in individuals in the control group and individuals in the influence group. Linkage disequilibrium between two populations for a given SNPs is used as an indication of the physical proximity to the genome between SNPs and genomic regions involved in drug response or disease susceptibility.

SNPs are the most common form of gene polymorphism. Combined with the potential as a functional variation, this has generated great interest as a gene mapping marker for mapping genes of pharmacogenetic indicators and complex diseases (Risch et al., 1996, Science 273: 1516-7; Kruglyak, 1997). Genet. 17: 21-4; Masood, 1999, Nature 398: 545-6). Many SNPs have already been identified, with> 2,500,000 in the NCBI's SNP database (http://www.ncbi.nlm.nih.gov/SNP/). Many recent studies have focused on identifying polymorphisms in the coding sequences of potential candidate genes associated with common diseases (Nickerson et al, Nat. Genet., 1998 19: 233-240; Cambien et al, 1999 , Am. J. Hum. Genet. 65: 183-91; Risch et al., 1996, Science 273: 1516-7; Kruglyak, 1997, Nat. Genet. 17: 21-4; Masood, 1999, Nature 398: 545-6; Cargill et al., 1999, Nat. Genet. 22: 231-238; Halushka et al., 1999, Nat. Genet. 22: 239-247).

In the state of the art, SNPs must first be discovered by intensively resequencing the genomes of large portions of individuals from as many as 100 well selected control groups. The most common differences found are candidates for SNPs. This approach is very time consuming and expensive, and the result depends on the choice of the control group.

Once SNPs have been identified, a rapid and economic way of evaluating the individual's numerous SNPs should be developed. In the state of the art, most methods of assessing SNPs rely on amplification by PCR (or other methods of DNA amplification) of small regions surrounding the SNP. This amplification step requires knowledge of the sequences surrounding the SNPs and the use of specific conventionally prepared nucleic acid primers for each SNP. Simultaneous amplification of many different DNA sequences is a tedious and expensive task, requiring complex and expensive robotics and large amounts of expensive reactants.

The ability to genotyping these large amounts of mutant material quickly and accurately has become an even more important goal in the genetic community (Bonn, D., 1999, Lancet, 353: 1684). The variety of techniques available has the potential to be used in highly efficient xenopipe laboratories (Landegren et al., 1998, Genome Research 8: 769-776). These techniques include 5 'exonuclease assays such as TaqMan (Lyvak et al., 1995, Nature Genet. 9: 341-342), molecular beacons (Tyagi et al, 1998, Nat. Biotechnol. 16 : 49-53), oligonucleotide-ligation assays (OLAs) (Tobe et al., 1996, Nucleic Acids Res. 24: 3728-3732), dye-labeled oligonucleotide ligation (DOL) (Chen et al., 1998, Genome Res., 8: 549-556), minisequencing (Chen et al., 1997, Nucleic Acids res., 25: 347-353; Pastinen et al., 1997, Genome Res. 7: 606 -614), microarray technology (Hacia et al., 1998, Genome Res. 8: 1245-1258; Wang et al., 1998, Science, 280: 1077-1082) and scorpions assay (Whitcome et al. , 1999, Nat. Biotechnol. 17: 804-807).

These existing methods have two major obstacles, one is that SNPs must be identified and chosen arbitrarily before evaluation, and the other is that numerous different DNA products must be produced by specific amplification methods. Therefore, we design a method that does not have these two drawbacks.

In the state of the art, existing methods do not meet the needs of the pharmaceutical industry. In addition to the pharmaceutical industry, many other fields, such as medical research, health care, veterinary medicine, agriculture, food, cosmetics, and many other industries and sectors, are interested in using the same approach based on different situations and / or different organisms. . Thus, there is a need for new methods for sufficient access to life's rich genetic changes, which are cheap and have very high efficiency with very high efficiency.

Thus, in order to identify faint genetic risk factors that were not detected in current genome scans due to the use of fewer markers, limited sample size, and / or sample pools, a more extensive analysis of variations in numerous sequences in a sample of many patients was performed. An effective method is needed. It is therefore an object of the present invention to provide a more efficient sequencing method.

No discussion or citation of references herein shall be construed as an admission that such references are prior art to the present invention.

The present invention relates to a method for detecting genome wide sequence variation associated with a phenotype in a species of organism. The invention also relates to a method for generating a genome wide restriction sequence tag for a living being.

1 shows a method for identifying restriction sequence tags associated with phenotypes.

2A and 2B show embodiments of the invention for determining restriction sequence tags.

3A and 3B show an embodiment of determining restriction sequence tags by generating restriction fragments from the genome of a living organism using restriction enzymes that cleave at both sides of the recognition site.

4A and 4B show an embodiment of determining restriction sequence tags by generating restriction fragments from the genome of living things using type IIS endonucleases.

5A and 5B show an embodiment of double digestion: determining restriction sequence tags by generating restriction fragments from the genome of living things using a rare cutter followed by the use of frequent cutters.

6A and 6B show an embodiment of double digestion: determining restriction sequence tags by generating restriction fragments from the genome of life using a first restriction enzyme and a plurality of second restriction enzymes.

7A and 7B show another embodiment of determining the restriction sequence tag by generating restriction fragments from the genome of the organism using digestion: first restriction enzyme and a plurality of second restriction enzymes.

8A and 8B show another embodiment of determining the restriction sequence tag by generating restriction fragments from the genome of the organism using digestion: the first restriction enzyme and a plurality of second restriction enzymes.

9A shows the generation of short DNA tags from cloned DNA fragments. Long DNA fragments are cloned between the BsmFI sites with annular vectors. BsmFI digestion leaves only a short DNA tag attached to the vector. After self ligation, the annular vector contains an insert formed by a pair of tags, regardless of the length of the original DNA fragment insert. 9B shows the analysis results of the product after the first ligation. Lambda phage DNA digested with Sau3AI was ligated with BamHI digested / dephosphorylated primary production vector. For analysis, the product was amplified by PCR using primers flanking the insertion site. 9C shows the analysis results of the product after the second ligation. The same samples as in FIG. 9B were normalized to their size by BsmFI decomposition and self ligation to generate annular vectors. For analysis, this product was amplified by PCR. An expected peak of 132 bp is observed. 9D shows the results of analysis of the second ligation product obtained in a simple reaction. Plasmids containing a single insert were treated with BsmFI and subjected to Klenow enzyme followed by self ligation to produce blunt ends. For analysis, the product was amplified by PCR. No bands were observed that correspond to fragments smaller than the correct size.

FIG. 10A shows several possibilities for DNA produced by digestion using two different enzymes to be cloned into a secondary production vector. 10B shows the results of in vitro cloning with secondary production vectors. MspI and SphI digested lambda DNA was inserted into the vector digested with SphI and AccI. After digestion with BsmFI, the second ligation produced inserts, restriction sequence tags of normalized size. PCR products obtained by amplification of the final ligation reaction were analyzed. Only bands of the correct size were observed. 10C shows the results of analysis of the original ligation products when AluI and SphI digested lambda DNA were inserted into the HincII and SphI digest vectors. After PCR amplification for analysis, fragments of different sizes were observed using the Agilent 2100 bioanalyzer DNA 1000 chip for analysis as expected. The highest peak is the size marker. FIG. 10D shows the analysis results of the same sample as in FIG. 10C after the second ligation. After PCR amplification, only a single fragment of expected size was observed using an Agilent 2100 bioanalyzer DNA 1000 chip. Peaks corresponding to the size markers are shown in the figure.

11A-11B show template preparation of HindIII and RsaI digested DNA using the single restriction sequence tag method shown in FIG. 4A. 11C shows the results of radiographic analysis of the collected aliquots after various steps of the method. Lane 1: PCR product of 350 bp of complete DNA colony vector size; Lanes 2-6: lambda genomic DNA and lanes 7-10 human genomic DNA; Lanes 3 and 7: after fragmentation to a short arm several fragments were observed; Lanes 4 and 8, size leveling after degradation with MmeI; Lanes 5, 6, 9, and 10: DNA colony vectors were generated at desired sizes after ligation into long cancers. 11D shows DNA colonies of lambda DNA. Figure 11E shows DNA colonies of lambda DNA (left column) or human DNA (first three images of right column). This DNA colony is then sequenced in situ using the method of WO 98/44152 to identify restriction sequence tags.

Figure 12 shows the generation of smooth ends from the 3 'overhang (description of PstI degradation) and partial filling of the 5' overhang with Klenow polymerase in the presence of dCTP (description of MspI degradation).

Summary of the Invention

The present invention provides a method for determining genome-wide sequence variation in a manner, preferably hypothesized, associated with a phenotype of a species. In one embodiment, genome-wide variation is determined from a subpopulation of individuals with a particular phenotype. In the methods of the invention, each individual's restriction fragment set in a subpopulation of individuals having a phenotype is generated by digesting the nucleic acid of the individual using one or more different restriction enzymes. Preferably, such sets of restriction fragments comprise a sufficient number of different restriction fragments to make it possible to identify sequence variations in the genome of an organism. More preferably, the restriction fragment set includes at least 10, 100, 1000, 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ different restriction fragments.

Then, a set of restriction fragment tags is determined for each individual from that restriction fragment set of the individual. In the method of the invention, a set of restriction sequence tags in an individual of a subpopulation with a particular phenotype preferably generates a set of restriction fragments, e.g., from the individual's genomic DNA, and then of each restriction fragment. Some are determined by sequencing using a method (described below) that involves the generation of DNA colonies.

Then, a set of restriction fragment tags obtained from different individuals of the sub-populations is preferably compared, so that each group is classified into one or more groups containing restriction sequence tags containing homologous sequences. Such a comparison preferably enables the determination of the number or frequency of each group of restriction sequence tags. Collection of homologous restriction tag groups of subpopulations can be used to identify sequence variations associated with phenotypes. In a preferred embodiment, the restriction sequence tag is compared with the genomic sequence of the organism to identify the genomic location of the restriction sequence tag. In another preferred embodiment, restriction sequence tags flanking both recognition sites are also identified from the genomic sequence of the organism.

Detailed description of the invention

The present invention provides a method for determining genome-wide sequence variation associated with a phenotype of a species (see, eg, FIG. 1). The present invention is based on the discovery that sequence variations associated with a phenotype can be determined by acquiring and comparing a sufficient number of sequence tags of genomic DNA or cDNA of an individual having that phenotype without hypothesis. For example, genome-wide variation can be determined from a subpopulation of individuals with a particular phenotype, such as individuals belonging to a particular race, variety, species, genus, family, etc. having the same phenotypic feature. Genome-wide variations can also be determined from subgroups, such as, for example, healthy individuals, individuals susceptible to certain diseases, or individuals in certain states of development.

In the methods of the invention, a set of restriction fragments for each member of a subpopulation of an individual with the phenotype is produced by digesting nucleic acids from the individual with one or more different restriction enzymes. As used herein, the restriction fragment set may include one or more restriction fragments. The set of restriction sequences tags of the individual is then determined from the set of restriction fragments. The restriction sequence tags of the organism subpopulation are compared and classified into one or more groups, each containing a restriction sequence tag comprising a homologous sequence. In one implementation, a restriction tag group consists of restriction tags with at least 60%, 70%, 80%, 90%, or 99% homologous. In another implementation, the restriction tag group consists of restriction tags that are 100% homologous. One or more restriction sequence tag groups obtained can be used to identify sequence variations associated with the phenotype. In a preferred embodiment, the phenotype under study relates to the proportion or combination of sequence variations. The present invention also provides a method for determining genome-wide sequence variation among multiple phenotypes by comparing restriction sequence tags of different phenotypes. The organism of the method of the present invention is applicable to any species. The method of the present invention is particularly useful for higher eukaryotes with complex genomes, such as but not limited to higher animals and plants, including humans. In particular, the methods of the invention are useful for analyzing and identifying sequence variations associated with response to disease susceptibility or treatment in humans.

The method of the present invention can be used to identify polymorphisms of the genome of a species from restriction sequence tags. The method does not have to find many sets of polymorphisms before initiating the correlation study compared to the conventional method; Ii) there is no need to select a limited set of polymorphisms before initiating correlation studies; Iii) there is no need to use prior knowledge of any sequence; Iii) no material to synthesize many sets of different oligonucleotides; Iii) no need to perform a large number of specific amplification steps; Iii) using a number of different restriction enzymes can easily increase the number of polymorphisms used in the study; Iii) the entire process is performed by manipulating a single physical sample, while in other methods there is at least one amplification step in which the number of physical samples is proportional to the number of polymorphs to be analyzed; Iii) there is no need to collect a sample of the population since each individual can be analyzed; Iii) identify sequence variations that exist at very low frequencies in a population; Iii) several advantages that the analysis cost is more than 10 times cheaper than current genotyping.

In the following description and examples, many terms are used. For a clear and consistent understanding of the specification and claims, the following definitions are provided.

The term “genomic region” refers to a portion of a genome containing one or multiple sequence variations, identified by comparing samples from a population of individuals using the methods of the invention.

The term “nucleic acid” refers to two or more nucleotides covalently linked to each other. Nucleic acids of the invention may contain phosphodiester bonds. Nucleic acids of the present invention are for example phosphoramide (e.g., Beaucage et al, 1993, Tetrahedron 491925, which is incorporated herein by reference in its entirety), phosphorothioate (e.g., incorporated herein by reference in its entirety) Mag et al, 1991, Nucleic Acids Res. 19: 1437 and US Pat. No. 5,644,048), phosphorodithioates (e.g. Briu et al. (1989) J. Am. Chem. Soc. 111: 2321), O- Methylphosphoramidate linkages (eg, Eckstein, Oligonucleotide and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (eg, Egholm (1992) J. Am. Nucleic acid analogues having a backbone including Chem. Soc. 114: 1895; Nielsen (1993) Nature 365: 566). Other analogue nucleic acids include positive backbones (e.g., Denpcy et al (1995) Proc. Natl. Acad. Sci. USA 92: 6097, which is hereby incorporated by reference in its entirety), nonionic backbones (herein incorporated by reference in their entirety). Nonribose skeletons, including those described in US 5,386,023; US 5,637,684; US 5,602,240; US 5,216,141; and US 4,469,863), and US 5,235,033 and US 5,034,506, which are incorporated herein by reference in their entirety. Nucleic acids comprising one or more carbocyclic sugars are also included within the definition of a nucleic acid (eg, Jenkins et al. (1995) Chem. Soc. Rev., 169-176, which is hereby incorporated by reference in its entirety). Several nucleic acid analogs are also described in Rawls, C & E News, June 2, 1997, 3, which is hereby incorporated by reference in its entirety. Such modifications of the ribose-phosphate backbone can be done to promote the addition of additional moieties such as labels or to increase the stability and half-life of such molecules within the physiological environment. It is also possible to prepare mixtures of naturally occurring nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs thereof. One of ordinary skill in the art would know how to select appropriate analogs for use in the various embodiments of the present invention. For example, natural nucleic acids are preferred when digested with restriction enzymes. Nucleic acids may be specified as single-stranded or double-stranded, or may contain portions of both double-stranded or single-stranded. Nucleic acids include, for example, DNA, such as genomic DNA, cDNA, RNA, or any combination of deoxyribo- and ribo-nucleotides and uracil, adenine, thymine, cytosine, guanine, inosine, xanthan, hypoxantanine, It may be a hybrid DNA containing any combination of bases including isocytosine, isoguanine and the like.

As used herein, the term "oligonucleotide" includes linear or oligomers with natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, and the like, and includes Watson-Crick type base pairing, base base stacks A specific pattern of monomer-to-monomer interactions, such as stacking, Hoogsteen or reverse Hoogsteen type base pairing, can be specifically bound to the target polynucleotide. Preferably, the monomers are linked by phosphodiester bonds or analogs thereof form oligonucleotides ranging in size from tens of monomer units (eg 40-60) from several (eg 3-4) monomer units. Whenever an oligonucleotide is represented by a letter sequence such as "ATGCCTG", the nucleotide is understood from 5 'to 3' direction from left to right, unless otherwise specified, "A" is adenine, "C" is cytidine, "G "Guanosine," T "represents thymidine, and" U "represents uridine. The term “nucleotide” refers to “deoxyribonucleotide” or “ribonucleotide,” and “dATP”, “dCTP”, “dGTP”, “dTTP”, and “dUTP” refer to triphosphate derivatives of each nucleotide. Usually, oligonucleotides include natural nucleotides, but they also include non-natural nucleotide analogues. It will be apparent to those skilled in the art that oligonucleotides composed of natural nucleotides are preferred, although, for example, it may be possible to apply oligonucleotides with natural or unnatural nucleotides when performing treatment with enzymes.

The term "polymorphism" refers to the presence of two or more alleles in a population. The term “allele” refers to one of several alternative sequence variations at a particular location. Polymorphisms at a single chromosome location constitute genetic markers. The term "SNP" refers to Single Nucleotide Polymorphism. Preferably, genetic variations, such as, for example, SNPs, are common in the population of organisms and are inherited in accordance with the Mendel trend. Such alleles may or may not be related to the phenotype.

As used herein, the term “heterozygote” refers to an individual with different alleles at corresponding positions on homologous chromosomes. Thus, the term “heterozygous” as used herein refers to an individual or strain having different alleles at one or more paired positions on homologous chromosomes.

The term “homozygote” as used herein refers to an individual with the same allele at the corresponding position on the homologous chromosome. Thus, the term "homozygous" as used herein refers to an individual or strain having the same allele at one or more paired positions on a homologous chromosome.

The term "variation" refers to a heritable change in the DNA sequence of an organism.

The term “genotype” is commonly known to refer to (i) the genetic makeup of the individual, or (ii) the type of allele found at the chromosomal location of the individual.

The term "limiting endonuclease" or "limiting enzyme" recognizes a particular base sequence (target or recognition site) in a double-stranded DNA molecule to direct the DNA molecule to, for example, within or near a target or recognition site. Refers to an enzyme that cleaves from

The term "restriction site" refers to, but is not limited to, usually 4-8 nucleotides, or 20 or more nucleotide sites in a nucleic acid, preferably a double-stranded nucleic acid, comprising a recognition site or cleavage site of a restriction endonuclease. Does not refer to a nucleotide site. The recognition site corresponds to a sequence within a nucleic acid to which a restriction endonuclease or restriction endonuclease group binds. The cleavage site or cleavage site corresponds to the specific sequence where cleavage by the restriction endonuclease occurs. Depending on the restriction endonuclease, the cleavage site may be within the recognition site. However, some restriction endonucleases, such as, for example, type-IIS endonucleases, have cleavage sites outside the recognition site.

The term "limiting fragment" refers to a DNA molecule produced by digesting a DNA molecule with restriction endonucleases.

The term “engineered nucleic acid” or “adaptor” refers to a short double-stranded DNA molecule having a predetermined nucleotide sequence. Preferably the engineered nucleic acid or adapter is 10 to 500 base pairs in length. More preferably, the engineered nucleic acid or adapter has a length of 10 to 150 base pairs. Preferably, it is defined in such a way that it can be ligated to the end of the restriction fragment. Such nucleic acids can be designed by those skilled in the art once given the sequence of restriction fragment ends. Preferably, the engineered nucleic acid comprises one or more amplification primer sequences, each of which is preferably oriented such that it is close to the end of the engineered nucleic acid and primer extension is enabled towards the end of the molecule. Amplification primers can be the same or different. Preferably, the engineered nucleic acid also comprises one or more sequencing primer sequences, each of which is oriented to allow primer extension in a direction close to the end of the engineered nucleic acid and towards the end of the molecule. Sequencing primers can be the same or different. In certain embodiments, the engineered nucleic acid can also include one or more restriction sites. Engineered nucleic acids are also referred to herein as DNA colony vectors.

The term "ligation" refers to an enzymatic reaction in which two double-stranded DNA molecules promoted by ligase are covalently bonded to each other. One or both DNA strands may be covalently bonded to each other. It is also possible to inhibit the ligation of either of the two strands through chemical and / or enzymatic modification of one of the ends to allow binding of only one of the two DNA strands.

The term "solid support" refers to any solid surface to which nucleic acids can be attached, such as but not limited to latex beads, dextran beads, polystyrene, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, and silicon wafers. Say Preferably, the solid support is a glass surface.

The term "nucleic acid colony" or "colony" refers to an isolated site, for example on a solid surface, that includes a plurality of copies of a nucleic acid strand. Multiple copies of complementary strands may also be present in the same colony. Multiple copies of the nucleic acid strands that make up the colony are generally immobilized on a solid support and may be in single or double stranded form.

As used herein, the term “colony primer” refers to a nucleic acid molecule comprising an oligonucleotide sequence capable of hybridizing to a complementary sequence to initiate a specific polymerase reaction. Sequences comprising colony primers are selected to have maximum hybridization activity with complementary sequences and very low nonspecific hybridization activity with any other sequence. Colony primers may have a length of 5 to 100 bases, but may preferably have a length of 15 to 25 bases. Naturally occurring or non-naturally occurring nucleotides may be present in the primer. One or more different colony primers can be used to generate nucleic acid colonies in the methods of the invention.

5.1 Collecting and recording samples from individuals of specific phenotype

Genomic DNA or cDNA of an individual with a particular phenotype can be derived from a sample collected from such individual. Preferably, for example, an individual belonging to a particular race, variety, species, genus, family, etc. having the same phenotypic characteristics, or having a particular condition, for example a healthy condition, a particular disease, or a particular stage of development. Identify subgroups of entities with the same phenotype as the subject. Collect samples of subpopulations of such individuals and record the phenotypic features associated with that subpopulation in detail. Such careful recording facilitates the assignment of sequence variations for one or more phenotypes.

5.2. How to generate restriction fragments by restriction decomposition

The method of the present invention comprises generating a set of restriction fragments from genomic DNA or cDNA of an organism such as, for example, genomic DNA extracted from a cell derived from an organism or cDNAs prepared from mRNAs extracted from a cell derived from an organism. Involve. In the present invention, DNA, eg genomic DNA, can be obtained from an individual, for example from different cells, parts, tissues, or organs. In various embodiments of the invention, one or more different restriction enzymes are used simultaneously or separately to generate a set of restriction fragments, for example from genomic DNA. Preferably, the restriction fragment set comprises a sufficiently large number of restriction fragments to make it possible to identify sequence variations in the genome of an organism. More preferably, the restriction fragment set includes at least 10, 100, 1000, 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , or 10 ⁸ different restriction fragments.

Nucleic acid molecules to be analyzed, such as genomic DNA, can be obtained from any source such as, for example, tissue homogenates, blood, amniotic fluid, chorionic villus samples, and bacterial cultures. The nucleic acid molecule can be obtained from such raw materials using conventional methods known in the art. Preferably only a very small amount of DNA or RNA nucleic acid is required (in the case of RNA a reverse transcription step is required before PCR). When molecular biological methods are used in the methods of the present invention, they are performed using standard methods (e.g. Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York 1989; Sambrook et al., Molecular Cloning, Laboratory). ^{Manual, 3 rd Editions, Cold Spring} Harbr New York, 2001; Innis et al, PCR Protocols:. AGuide to Methods and Applications, Academic Press, Cold Spring Harbor New York, 1989).

Any restriction enzyme known in the art can be used in the present invention. In certain embodiments of the invention, Type-IIS endonucleases are generally commercially available and are well known in the art. Type-IIS endonucleases recognize specific sites of base pairs within double-stranded polynucleotide sequences. Upon recognition of the sequence, the endonuclease will cleave the polynucleotide sequence, leaving one strand of that sequence with a longer “sticky end”. Type-IIS endonucleases, like type-II endonucleases, must have a specific recognition site palindrome, ie the base pair sequence is identical on both strands of the recognition site when read in the 5 'to 3' direction. Do not require to do In addition, Type-IIS endonucleases also cleave outside of the recognition site. Because cleavage occurs at the location of any polynucleotide sequence that is a given base pair away from the recognition site, Type-IIS makes it possible to capture an intervene sequence between the cleavage sites in some embodiments of the invention. . Type-specific ⅡS endonuclease useful in the present invention Ear I. Mnl I, Ple I, Alw I, Bbs I, Bce AI, Bsa I, Bsm AI, Bsp MI, Eco 571, Esp 3I, Hga I, Sap I, Sfa NI, Bbv I, Bsm FI, Fok I, Bse RI, Hph I, Mme I, and Mbo IL, but are not limited thereto. The enzyme currently found cleaves up to 20-25 bases from its recognition site. For example, enzymes that cleave sites 50, 100, or 200 bases away from the recognition site will be useful in the present invention.

In some embodiments of the invention, a combination of rare and frequent cutters is used to create the restriction fragment. The rare cutter is a restriction endonuclease having a recognition site consisting of at least 4 nucleotide sequences, preferably 6 or 8 nucleotide sequences. Examples of rare commercially available cutters are Pst I, Hpa II, Msp I, Cla I, Hha I, EcoR II, Bst BI, Hin PI, Mae II, Bbv I, Pvu II, Xma I, Sma I, Nci I, Ava I, Hae II, Sal I, Xho I, and Pvu II, among which Pst I, Hpa II, Msp I, Cla I, Hha I, EcoR II, Bst BI, Hin PI, and Mae II desirable. Frequent cutters are restriction endonucleases with up to 4 base nucleotide recognition sites. Examples of suitable frequent cutter enzymes include Mse I and Taq I.

In certain embodiments of the invention, the restriction fragment is linked to another nucleic acid or to itself at the site of degradation. Typically, restriction enzymes produce blunt ends where the base nucleotides of both strands are base paired, or a staggered end where one of the two strands protrudes, resulting in a short single strand extension. In certain embodiments of the invention, when the restriction enzyme is Type-IIS, it is preferred to add a step comprising modification of the ends by converting the protruding ends into smooth ends with polymerase.

5.3. How to Determine Restriction Sequence Tags

Any method known in the art can be used to determine the restriction sequence tag set of restriction fragments generated by the method of section 5.2. Preferably, the restriction fragments are amplified before sequencing. However, sequencing methods that do not require amplification, such as single molecule sequencing, can be used without additional amplification steps. Preferably, the resulting restriction sequence tag is at least 5 nucleotides in length. More preferably, the resulting restriction sequence tag is in the range of 10 to 20 nucleotides in length. More preferably, the resulting restriction sequence tag is 50 or less nucleotides in length. Preferably, a method comprising the generation and sequencing of DNA colonies is used to determine the restriction sequence tag of the restriction fragment. Any method known in the art can be used in the present invention (eg, PCT Publications WO 98/44151, WO 98/44152, WO00 / 18957, and WO 02/46456, which are hereby incorporated by reference in their entirety). ). One nucleic acid colony may be generated from a single immobilized nucleic acid template, eg, a nucleic acid template derived from a restriction fragment. The method of the invention also makes it possible to produce many such nucleic acid colonies simultaneously, each containing a different immobilized nucleic acid.

DNA colonies can be generated in a method comprising capturing and amplifying DNA fragments, eg, restriction fragments, using primers immobilized on a solid surface (see PCT Publications WO 98/44151 and WO 98/44152). In embodiments of the invention where the DNA fragment is annular, it is preferred to perform the step of linearizing the annular DNA fragment using restriction enzymes prior to colony production. In one embodiment, the DNA colony is

Iii) providing a solid surface comprising a plurality of colony primers immobilized at the 5 'end on the solid surface, wherein each colony primer comprises a sequence capable of hybridizing with the sequence at the 3' end of the DNA molecule of the sample. Characterized by;

Ii) denaturing said DNA molecule to produce a single stranded fragment;

Iii) annealing the single stranded fragment to the immobilized colony primer;

Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment;

Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments;

Iii) annealing the fixed single stranded fragments to the fixed colony primers;

Iii) repeating steps iii) to iii) such that the colonies are produced at each particular site on the solid surface, resulting from a sample of DNA molecule, for example a pool of restriction fragments.

In a preferred embodiment, the immobilized primer comprises a sequence capable of hybridizing with the sequence of the DNA molecule. For example, the DNA molecule in the sample may be a restriction fragment linked to a nucleic acid having a predetermined sequence. In such cases, the immobilized primer may have a sequence that is hybridizable to the sequence in the predetermined sequence. In other embodiments of the invention, colony primers having different sequences can be used. Primers for use in the present invention preferably have a length of at least 5 bases. More preferably, the primers have a length less than 100 or less than 50 bases. The present invention repeats the steps of annealing the template to the immobilized primer, extending the primer, and separating the extended primer from the template. . It will be appreciated by those skilled in the art that this step can be performed using PCR reagents and conditions (or reverse transcription and PCR). PCR techniques are disclosed, for example, in "Clinical Diagnostics and Research" published in 1992 by Springer-Verlag, which is hereby incorporated by reference in its entirety.

DNA colonies can also be produced by the methods described in PCT Publication WO 00/18957. In embodiments of the invention where the DNA fragments to be amplified are cyclic, it is preferred to perform the linearization of the cyclic DNA fragments with restriction enzymes prior to colony production. In one embodiment, the DNA colony is derived from a sample of DNA molecules, for example a restriction fragment pool,

Iii) mixing the DNA molecules in the sample with colony primers comprising sequences in which each colony primer hybridizes with the sequence at the 3 'end of the DNA molecule;

Ii) implanting DNA molecules and colony primers on the solid surface to the solid surface at the 5 'end of both the DNA molecules and colony primers to produce fixed linear fragments and fixed colony primers;

Iii) denaturing the immobilized DNA molecules to produce immobilized single stranded fragments;

Iii) annealing the fixed single stranded fragment to the fixed colony primer to obtain an annealed single stranded fragment;

Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment;

Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments;

Iii) annealing the fixed single stranded fragments to the fixed colony primers;

Iii) repeating steps iii) to iii) to produce colonies at each particular site on the solid surface.

Preferably, the proportion of colony primers present in the mixture is higher than the proportion of colony templates. Preferably, the ratio of colony primer to colony template comprises a plurality of colony primers that are located at appropriate uniform density over the entire or defined portion of the solid support when the colony primer and nucleic acid template are immobilized on the solid support. A "lawn" of solid colony primers is formed and one or more colony templates within the colon of the colony primers are spaced apart individually. Primers for use in the present invention preferably have a length of at least 5 bases. More preferably, the primers have a length of less than 100 or less than 50 bases. The present invention repeats the steps of annealing the template to the immobilized primer, extending the primer, and separating the extended primer from the template. It will be appreciated by those skilled in the art that this step can be performed using PCR reagents and conditions (or reverse transcription and PCR). PCR techniques are disclosed, for example, in "Clinical Diagnostics and Research" published in 1992 by Springer-Verlag, which is hereby incorporated by reference in its entirety.

Isothermal amplification of nucleic acids on solid supports can also be used to generate DNA colonies (see, eg, PCT Publication WO 02/46456). In embodiments of the invention where the DNA fragment to be amplified is annular, it is preferred to perform the linearization of the linear DNA fragment with restriction enzymes prior to colony production. In one embodiment, the DNA colony is derived from a DNA molecule sample, eg, a restriction fragment pool.

Iii) DNA molecules in the sample are mixed with colony primers, each colony primer comprising a sequence capable of hybridizing with the sequence at the 3 'end of the DNA molecule, wherein the concentration of the colony primer is amplified with the transplanted DNA molecule. Adjusting it to be;

Ii) grafting the DNA molecule and colony primer to the 5 'end on the solid surface to produce immobilized DNA molecule and immobilized colony primer;

Iii) applying an amplification solution containing polymerase and nucleotides to the solid surface such that the colonies are isothermally generated at each particular location of the solid surface.

The amount of nucleic acid immobilized in step ii) determines the average number of DNA colonies per surface that can be produced. The preferred concentration range of DNA molecules to be immobilized is preferably from 1 nanomolar to 0.01 nanomolar with colony template and from 50 to 1000 nanomolar with colony primer. In a preferred embodiment, the temperature of the reaction is selected as the optimum temperature of the polymerase activity. In a preferred embodiment, the DNA molecules in the sample have a size in the range of about 50-5000 base pairs.

In the method described in this section, colonies are produced at locations remote from each other on the surface. Colony density on the surface can be adjusted, for example, by adjusting the density of primers immobilized on the surface. In a preferred embodiment, the colony density is at least 10 ^4-6 colonies / cm ² , more preferably at least 10 ^7-8 colonies / cm ² . The size of the colonies can also be adjusted by adjusting the experimental conditions. Preferably, the colonies have a maximum diameter of 10 nm to 100 μm, more preferably 100 nm to 10 μm.

DNA colonies can be sequenced to determine at least a portion of the sequence of DNA. In one embodiment, sequencing is performed by hybridizing appropriate primers, sometimes referred to herein as “sequencing primers,” with the nucleic acid molecules of the DNA colony, extending the primers, and detecting the nucleotides used to extend the primers. Preferably, the nucleotides used to extend the primers in each colony are detected prior to adding the next nucleotide to the growing nucleic acid chain, making it possible to sequence the bases one by one in situ nucleic acid.

Detecting the integrated nucleotides is facilitated by including one or more labeled nucleotides in the primer extension. Any suitable detectable label can be used, for example fluorophores, radioactive labels and the like. Preferably fluorescent labels are used. Any fluorescent label known in the art can be used. The same or different fluorescent labels can be used for each different kind of nucleotide. If the label is a fluorophore and the same label is used for different kinds of nucleotides, each nucleotide integration provides a cumulative increase in the signal detected at a particular wavelength. If different labels are used, these signals can be detected at different suitable wavelengths. In a preferred embodiment, a mixture of nucleotides labeled and unlabeled nucleotides of the same kind is used in each primer extension step.

In order to be able to hybridize the appropriate sequencing primer to the nucleic acid template to be sequenced, the nucleic acid template should usually be in single stranded form. If the nucleic acid templates constituting the nucleic acid colony are in double stranded form, methods well known in the art, such as, but not limited to, denaturation, cleavage, etc., can be used to provide a single stranded nucleic acid template.

Sequencing primers that are hybridized to the nucleic acid template and used for primer extension are preferably short oligonucleotides, for example 15-25 nucleotides in length. The sequence of the primer can be designed to hybridize with a portion of the nucleic acid template to be sequenced under stringent conditions. The sequence of the primers used for sequencing may have the same or similar sequence as the sequence of colony primers used to generate nucleic acid colonies.

Adding nucleic acid templates and sequencing primers to appropriate conditions as determined by methods well known in the art, once sequencing primers are annealed to the nucleic acid template to be sequenced, for example, nucleic acid polymerase and at least a portion of the nucleotides in labeled form are provided. If appropriate nucleotides are provided, primer extension is performed using conditions appropriate for primer extension. DNA polymerases and nucleotides that can be used are well known in the art.

Preferably, after each primer extension step, a wash step is included to remove unintegrated nucleotides that may interfere with subsequent steps. After the primer extension step is performed, DNA colonies can be detected to determine whether the labeled nucleotides have been incorporated into the extended primer. The primer extension step can then be repeated to determine the nucleotides after integration into the extended primers.

Any device that allows detection of the presence or absence, and the amount of appropriate label incorporated into the extended primer, such as fluorescence and radioactivity, may be used for sequencing. In embodiments where the label is a fluorescent label, one can use a CCD camera attached to a magnifying device (eg, a microscope).

The detection system is preferably used with an assay system to determine the number and identity of nucleotides incorporated into each colony after each primer extension step. This analysis, which can be performed using data recorded immediately after or after each primer extension step, allows for the determination of the sequence of the nucleic acid template within a given colony.

In another embodiment of the invention, the entire sequence or partial sequence of one or more nucleic acids can be determined by determining the entire or partial sequence of a nucleic acid template present in one or more nucleic acid colonies. Preferably, multiple sequences are determined simultaneously and the nucleotides applied to the nucleic acid colonies are generally applied in the order selected, for example dATP, dTTP, dCTP, dGTP, and then repeated throughout the analysis.

Thus, it can be seen that the whole or partial sequence of the nucleic acid template constituting a particular nucleic acid colony can be determined.

Primers and oligonucleotides used in the methods of the present invention are preferably DNA, can be synthesized using standard techniques, and are detectably labeled using standard methods when appropriate (Ausubel et al., Supra). Detectable labels that can be used in the methods of the present invention include, but are not limited to, fluorescent labels (eg, fluorescein and damin). Labels used in the methods of the present invention are detected using standard methods.

The method of the present invention can be facilitated by using a kit containing the reagents needed to perform the assay. The kit may contain reagents for performing analysis of a single restriction fragment tag (eg for use in diagnostic methods) or analysis of several restriction fragment tags (eg for use in genomic mapping). When analyzing several samples, several sets of appropriate primers and oligonucleotides are provided in the kit. In addition, in addition to the primers and oligonucleotides required to perform the various methods, the kit may contain enzymes used in the methods, reagents for detecting labels, and the like. The kit may also contain a solid substrate for use in carrying out the method of the present invention. For example, the kit may contain a glass plate or a solid substrate such as silicon or glass microchips.

5.4. How to identify restriction sequence tags associated with phenotypes

The restriction sequence tags obtained from each individual are then compared between subpopulations of a given phenotype to identify all homologous tags and determine the number of homologous restriction sequence tags. In a preferred embodiment, two restriction sequence tags obtained within the DNA colony indicate the ends of the corresponding restriction fragment in the restriction fragment set. Such two tags originate from locations physically close to each other on the genome. Each tag can be combined with the sequence of a restriction site of a restriction enzyme that is used for digestion of genomic DNA to obtain longer sequences. Classify homologous tags. In one embodiment, the restriction tag group consists of restriction tags with at least 60%, 70%, 80%, 90%, or 99% homology. In another implementation, the unique restriction tag group consists of restriction tags that are 100% homologous. Collection of restriction tag groups of subpopulations can be used to identify sequence variations associated with phenotypes. In a preferred embodiment, the phenotype under study relates to the proportion of sequence variation in the population or to a combination of sequence variations. In one embodiment, for example, represented by the relative number of restriction tags in each one or more population specific groups of restriction sequence tags, each 10%, 20%, 50%, 70%, or 90% between two different populations. As described above, the proportion of one or more specific sequences in a population is identified as being associated with the phenotypic difference between the two groups. In another preferred embodiment, the phenotype is associated with a particular combination of sequence variations found in a population of individuals. In one embodiment, a combination of ratios of a plurality of specific sequences in a population, for example represented by a combination of the number of restriction tags in a plurality of specific groups of restriction sequence tags, ie, the total number of restriction tags in the plurality of groups, If such a combination of ratios differs by more than 10%, 20%, 50%, 70%, or 90% between the two groups, it is found to be associated with the difference in phenotype between the two groups. In another embodiment, many such combinations are used to identify differences in phenotypes. In embodiments where multiple combinations are used, each combination of multiple combinations may include one or more specific sequences that are included in different combinations in the multiple combinations. This embodiment is illustrated in Example 6.3 below.

In one embodiment, the restriction sequence tag may be compared with the genomic sequence of the organism to identify the genomic location of the restriction sequence tag. In another embodiment, restriction sequence tags flanking the genome on both sides of the recognition site are identified from the genetic sequence of the organism.

5.5. Certain Preferred Embodiments for Obtaining Restriction Sequence Tags

Several preferred embodiments for obtaining restriction sequence tags are described in this section. These methods can be prepared in combination with any of the methods described in Sections 5.1 to 5.4 to identify sequence variations associated with phenotypes. It will be apparent to those skilled in the art that any repetition and / or combination of one or more specific embodiments described in this section may also be used.

(1) First Specific Embodiment

In a preferred embodiment, the present invention provides a method for generating a restriction sequence tag of a biological sample (FIGS. 2A and 2B). In that method, one or more first restriction enzymes can be used to digest the nucleic acid extracted from the biological sample to generate a set of restriction fragments. after that,

1) linking a restriction fragment in a set of restriction fragments with a first engineered nucleic acid comprising a predetermined nucleotide sequence to obtain a first annular nucleic acid fragment, wherein the predetermined nucleotide sequence is determined by a second restriction enzyme by the restriction fragment; And at least one recognition site of the second restriction enzyme, positioned and oriented to cleave at;

2) digesting the first cyclic nucleic acid fragment with a second restriction enzyme;

3) modifying the terminal produced by the second restriction enzyme to enable ligation;

4) linking the ends produced by the second restriction enzyme to generate a second set of circular nucleic acid fragments; And

5) determining a restriction sequence tag set from the restriction fragment set using a method comprising sequencing at least a portion of each restriction fragment of the second circular nucleic acid to determine the restriction sequence tag set.

Preferably, each of the recognition sites of the second restriction enzyme of the first engineered nucleic acid is located close to the end of the first engineered nucleic acid. In one preferred embodiment, each of the second restriction enzyme recognition sites in the first engineered nucleic acid is located less than 20 nucleotides away from the end of the first engineered nucleic acid. More preferably, each recognition site of the second restriction enzyme of the first engineered nucleic acid is located 0 to 5 nucleotides away from the end of the first engineered nucleic acid. Preferably, the second restriction enzyme is a type IIS endonuclease. In a preferred embodiment, the type IIS endonuclease cleaves a base at least 5, 10, 20, 50, 100, or 200 away from the recognition site. In another embodiment, the second circular nucleic acid fragment can be linearized using, for example, a third restriction enzyme different from the first restriction enzyme and the second restriction enzyme to obtain a third set of restriction fragments. In a preferred embodiment, the method also includes amplifying the third restriction fragment using the primer found in the first engineered nucleic acid. In another preferred embodiment, digesting with a third restriction enzyme and then amplifying may be substituted for amplifying the second annular nucleic acid fragment.

In a preferred embodiment, the step of fixing and amplifying the second circular nucleic acid fragment is performed before step 5). In a more preferred embodiment, the fixation and amplification is performed by any one of the DNA colony methods described in section 5.3. In a more preferred embodiment, sequencing is performed by one of the base-by-base primer extension methods described in section 5.3.

In another more preferred embodiment of the invention, the step of modifying said end of said second restriction fragment is removed by supplementing with DNA polymerase or removing protruding nucleotides to blunt to connect the ends of said second restriction fragment. do.

In another preferred embodiment, the methods of the present invention comprise a purification step and / or a DNA separation step after each step.

In another preferred embodiment, the small genomic DNA sequence in the restriction fragment set is linked to the bacterium using a plasmid isolated from each individual bacterial colony and bacteria spread to some extent, inserted into the plasmid, and plated on agarose plates. Clones and sequence using Sanger sequencing into an automated capillary sequence. Other approaches that do not utilize bacterial cloning are also known to those of ordinary skill in the art. For example, the first engineered nucleic acid can include a combinatorial sequence tag such that the third nucleic acid fragment can be used for molecular cloning on the beads to sequence by one base.

(II) Second Specific Embodiment

In another embodiment, the present invention provides a method of generating a restriction sequence tag of a biological sample (FIGS. 3A and 3B). In this method, a nucleic acid extracted from a biological sample can be digested using a first restriction enzyme to generate a set of restriction fragments. The first restriction enzyme cleaves both recognition sites in such a way that the cleavage site, rather than the recognition site, surrounds part of the sequence. Restriction enzymes can be used for this purpose, including but not limited to Bae I, Bcg I, Bsa XI. after that,

1) modifying the terminus produced by the first restriction enzyme to enable ligation;

2) linking the restriction fragments in the restriction fragment set with a first engineered nucleic acid comprising a predetermined nucleotide sequence to obtain a first set of circular nucleic acid fragments; And

3) determining the restriction sequence tag set from the restriction fragment set by a method comprising sequencing at least a portion of each restriction fragment of the first circular nucleic acid to determine the restriction sequence tag set.

In a preferred embodiment, the step of fixing and amplifying the first circular nucleic acid fragments is performed before step 3). In a more preferred embodiment, the fixation and amplification is performed by any one of the DNA colony methods described in section 5.3. In a more preferred embodiment, sequencing is performed by the primer extension method by one base described in section 5.3.

In another preferred embodiment, the step of modifying said terminus of said second restriction fragment is carried out by supplementing with DNA polymerase to remove blunt nucleotides or by eliminating protruding nucleotides to connect termini.

In another preferred embodiment, the method of the present invention comprises a purification step and / or a DNA separation step after each step.

(III) Third Specific Embodiment

In another embodiment, the present invention provides a method of generating a restriction sequence tag of a biological sample (FIGS. 4A and 4B). In this method, a nucleic acid extracted from a biological sample can be digested using a first restriction enzyme to generate a set of restriction fragments. after that,

1) linking a restriction fragment in the restriction fragment set with a first engineered nucleic acid comprising a predetermined nucleotide sequence to obtain a first nucleic acid fragment set, wherein the predetermined nucleotide sequence is determined by a second restriction enzyme. At least one recognition site of a second restriction enzyme, positioned and oriented to cleave at the fragment;

2) digesting the first nucleic acid fragment with a second restriction enzyme;

3) altering the termini produced by the second restriction enzyme to enable ligation;

4) linking the terminus produced by the second restriction enzyme with a second engineered nucleic acid comprising a predetermined nucleotide to generate a second set of nucleic acid fragments; And

5) determining the restriction sequence tag set from the restriction fragment set by a method comprising sequencing at least a portion of each restriction fragment in the second nucleic acid fragment to determine the restriction sequence tag set.

Preferably, the recognition site of the second restriction enzyme in the first engineered nucleic acid is located close to the end of the first engineered nucleic acid. In one preferred embodiment, the recognition sites of the second restriction enzymes in the first engineered nucleic acid are located by less than 20 nucleotides apart. More preferably, each recognition site of the second restriction enzyme of the first engineered nucleic acid is located 0 to 5 nucleotides away from the end of the first engineered nucleic acid. Preferably, the second restriction enzyme is a type IIS endonuclease. In a preferred embodiment, the type IIS endonuclease cleaves at least 5, 10, 20, 50, 100, or 200 bases away from the recognition site.

In a preferred embodiment, the step of fixing and amplifying the second nucleic acid fragment is performed before step 5). In a more preferred embodiment, the fixation and amplification is performed by any one of the DNA colony methods described in section 5.3. In a more preferred embodiment, sequencing is performed by one of the base-by-base primer extension methods described in section 5.3.

In another preferred embodiment of the invention, the end of said second restriction fragment is removed by supplementing with DNA polymerase or removing protruding nucleotides to blunt to allow the step of altering the end of said second restriction fragment to be linked. Follow the steps to change it.

In another preferred embodiment, the methods of the present invention comprise a purification step and / or a DNA separation step after each step.

(IV) fourth specific embodiment

In another embodiment, the present invention provides a method of generating a restriction sequence tag of a biological sample (FIGS. 5A and 5B). In this method, one or more rare cutters are used to digest nucleic acid extracted from a biological sample to generate a set of restriction fragments. Preferably, a rare cutter is used that recognizes 6-base, 8-base, or more than 8-base recognition sequences. after that,

1) linking a restriction fragment in the restriction fragment set with a first engineered nucleic acid comprising a predetermined nucleotide sequence to obtain a first nucleic acid fragment set;

2) digesting the first nucleic acid fragment with a second restriction enzyme that is different from the first restriction enzyme without cleavage from the first engineered nucleic acid to produce a second restriction fragment;

3) linking the end of the second restriction fragment with a second engineered nucleic acid comprising a predetermined nucleotide sequence to generate a second set of nucleic acid fragments;

4) determining the restriction sequence tag set from the restriction fragment set by a method comprising sequencing at least a portion of each restriction fragment in the second nucleic acid fragment to determine the restriction sequence tag set.

In a preferred embodiment, digestion with the first restriction enzyme and the second restriction enzyme is performed simultaneously prior to ligation with the first and second engineered fragments.

In a preferred embodiment, the step of fixing and amplifying the second nucleic acid fragment is performed before step 4). In a more preferred embodiment, the fixation and amplification is performed by any one of the DNA colony methods described in section 5.3. In a more preferred embodiment, sequencing is performed by one of the base-by-base primer extension methods described in section 5.3.

In another preferred embodiment, the methods of the present invention comprise a purification step and / or a DNA separation step after each step.

(V) other specific embodiments

The invention also provides a method of generating a restriction sequence tag of a biological sample. In such a method, a set of restriction fragments is generated using one or more first restriction enzymes to degrade nucleic acid extracted from a biological sample. Then, the restriction fragment is further digested using a plurality of different second restriction enzymes. Such a method makes it possible to further increase the number of restriction sequence tags located close to the recognition site of the first restriction enzyme.

In a preferred embodiment (FIGS. 6A and 6B),

1) linking a restriction fragment in the restriction fragment set with a first engineered nucleic acid comprising a predetermined nucleotide sequence to obtain a first nucleic acid fragment set;

2) digesting the first nucleic acid fragment with a second restriction enzyme that is different from the first restriction enzyme without cleavage from the first engineered nucleic acid to obtain a second restriction fragment;

3) linking the end of the second restriction fragment with a second engineered nucleic acid comprising a predetermined nucleotide sequence to generate a second set of nucleic acid fragments;

4) determining the restriction sequence tag set from the restriction fragment set after digestion with the first restriction enzyme, by sequencing at least a portion of each restriction fragment in the second nucleic acid fragment to determine the restriction sequence tag set. do.

In another preferred embodiment (FIGS. 7A and 7B), after digestion with a first restriction enzyme,

1) The restriction fragment in the restriction fragment set, wherein the second restriction enzyme and the third restriction enzyme include two recognition sites of different restriction enzymes and two recognition sites of the third restriction enzyme and the recognition sites of the second restriction enzyme Is a first engineered nucleic acid comprising a predetermined nucleotide sequence located between the recognition sites of the third restriction enzyme and the recognition site of the third restriction enzyme is located and orientated such that the third restriction enzyme is cleaved at the restriction fragment. Concatenating with to obtain a first set of circular nucleic acid fragments;

2) digesting the first nucleic acid fragment with a second restriction enzyme to obtain a second nucleic acid fragment;

3) joining the ends of the second restriction fragment to generate a second set of circular nucleic acid fragments;

4) determining a set of restriction sequence tags from the set of restriction fragments by a method comprising sequencing a portion of each of the restriction fragments of the third circular nucleic acid fragment to determine the set of restriction sequence tags.

Preferably, the method further comprises, after step 3): 3i) digesting the second circular nucleic acid fragment with a third restriction enzyme to generate a third set of nucleic acid fragments; 3ii) modifying the termini produced by the third restriction enzyme to enable ligation; And 3iii) linking the ends of the third nucleic acid fragments to generate a third set of circular nucleic acid fragments. Preferably, the recognition site of the third restriction enzyme in the first engineered nucleic acid is located close to the end of the first engineered nucleic acid. In one preferred embodiment, each of the recognition sites of the third restriction enzyme in the first engineered nucleic acid is located less than 20 nucleotides apart. In a more preferred embodiment, each recognition site of the third restriction enzyme of the first engineered nucleic acid is located 0 to 5 nucleotides away from the end of the first engineered nucleic acid. Preferably, the third restriction enzyme is a type IIS endonuclease. In a preferred embodiment, the type IIS endonuclease cleaves at least 5, 10, 20, 50, 100, or 200 bases away from the recognition site.

In a more preferred embodiment (FIGS. 8A and 8B), after digestion with a first restriction enzyme,

1) The restriction fragment in the restriction fragment set is linked with a first engineered nucleic acid comprising a predetermined nucleotide sequence comprising a recognition site of a second restriction enzyme different from the first restriction enzyme to link the first nucleic acid fragment set. Obtaining;

2) digesting the first nucleic acid fragment with a second restriction enzyme to obtain a second restriction fragment;

3) joining the ends of the second restriction fragment to generate a first set of circular nucleic acid fragments; And

4) The restriction sequence tag set is determined from the restriction fragment set after digestion with the first restriction enzyme by a method comprising sequencing at least a portion of each of the fourth nucleic acid fragments to determine the restriction sequence tag set. Preferably, the method further comprises, after step 3): 3i) digesting the first cyclic nucleic acid fragment with a third restriction enzyme different from the first and second restriction enzymes to generate a third set of nucleic acid fragments; 3ii) modifying the termini produced by the third restriction enzyme to enable ligation; And 3iii) linking the ends of the third nucleic acid fragments to generate a third set of circular nucleic acid fragments.

In such embodiments, it is desirable to further digest separately the set of restriction fragments generated by the first restriction enzyme using each of a plurality of different second restriction enzymes. More preferably, the plurality of different second restriction enzymes comprise at least three, five, ten, or twenty different restriction enzymes.

In a preferred embodiment, the step of fixing and amplifying the first nucleic acid fragment is performed before the sequencing step. In a more preferred embodiment, the fixation and amplification is performed by any one of the DNA colony methods described in section 5.3. In a more preferred embodiment, sequencing is performed by the primer extension by one base described in section 5.3.

In another preferred embodiment, the step of altering said terminus of said second restriction fragment is carried out by supplementing with DNA polymerase to remove blunting or by removing protruding nucleotides so as to be linked.

In another preferred embodiment, the methods of the present invention comprise a purification step and / or a DNA separation step after each step.

Such an embodiment makes it possible to identify two restriction sequence tags included in each first restriction fragment part, wherein the first restriction tag is next to the first restriction enzyme recognition site and the second restriction tag Is next to the second restriction enzyme recognition site, which stores the information that the first and second restriction sequence tags are paired restriction sequence tags derived from the same first restriction fragment.

Restriction sequence tags can be classified by sequence homology, possibly further classifying a pair of restriction sequence tags containing the same first restriction sequence tag, and a second restriction from the sorted paired restriction sequence tag The tag stores information that is located on the same physical plane or on the same side as the given first restriction enzyme recognition site. In a preferred method of the invention, if genomic sequences are available, the addition of clustering restriction sequence tags by mapping to identify flanking restriction sequence tags located on the genome present on both sides of the recognition site of the first restriction enzyme. Provides an important step

6. Examples

The following examples are intended to illustrate the invention, but not to limit the invention in any way.

6.1. Example 1 Preparation of DNA Colony Templates: Double Restriction Sequence Tag

This example illustrates the manipulation of a vector for the in vitro generation of DNA tags. An embodiment of the generation of restriction sequence tags from genomic DNA is shown in FIG. 9A. This example uses a plasmid vector with a DNA cloning site located between two BsmFI sites. The vector is based on the selected pUC19 plasmid due to its small size.

1) First Generation of Cloning Vectors

The primary generation of cloning vectors was designed for use with genomic DNA digested with a single restriction enzyme. In this example, bacteriophage lambda genomic DNA was used to demonstrate the generation of restriction sequence tags.

Two vector variants were made by cloning the synthetic linker with pUC19. In a first variant, the vector contains the following inserts:

BsmFI BamHI BsmFI

GGGAC GGATCC GTCCC (SEQ ID NO: 1)

CCCTG CCTAGG CAGGG (SEQ ID NO: 2)

This makes it possible to clone Sau3AI digested lambda DNA into BamHI restriction sites flanked by two BsmFI sites. The BamHI site of pUC19 was previously removed from the vector.

In a second variant, the vector contains an insert with an AatII restriction site (underlined) formed by two adjacent BsmFI sites.

BsmFI BsmFI

GG GAC GTC CC (SEQ ID NO: 3)

CC CTG CAG GG (SEQ ID NO: 4)

AatⅡ

This makes it possible to clone TaiI digested lambda DNA into vectors. The AatII site of pUC19 was previously removed from the vector.

Both primarys were dephosphorylated before use to prevent self ligation of empty vectors. After ligation of lambda DNA fragments, DNA polymerase I and ligation were used to restore the integrity of the DNA strands.

The following summarizes the steps (commonly used for another generation of vectors):

Iii) Ligation 1

Ii) inactivation of T4 DNA ligase by heating

Iii) decomposition into BsmFI

Iii) DNA termination supplementation with Klenow and dNTPs

Viii) deactivation of BsmFI by heating;

Iii) second ligation reaction

The following in vitro restriction sequence tag generation protocol was used in the examples:

Primary Ligation

0.1 μg bacteriophage lambda genomic DNA digested by appropriate enzymes

0.05 μg linear vector (purified by agarose gel)

1 mM ATP

1-x Buffer NEB4

1 μl T4 DNA ligase (New England Biolabs, 400 u / μl)

10 μl total volume, 2 hours incubation at room temperature

Inactivation by heating T4 DNA ligase at 65 ° C. for 20 minutes

BsmFI Decomposition

1-x buffer NEB4,

Add 5 μl of solution containing 0.5 μl BsmF1 (New England Biolabs, 2 u / μl)

2 hours incubation at 65 ℃

Klenow Treatment

1-x buffer for T4 DNA ligase (New England Biolabs),

100 μM dNTPs,

Add 5 μl of solution containing 0.5 μl Klenow fragment (New England Biolabs, 5 u / μl)

Incubate for 5 minutes at room temperature

Inactivate enzyme by heating at 80 ° C. for 20 minutes

Secondary Licensing

1-x MSL buffer,

2 mM ATP

20% PEG6000

Add equal volume of solution containing 10% (v / v) T4 DNA ligase (New England Biolabs, 400 u / μl)

Incubate overnight at 16 ℃

In this protocol, Minimal Salt Ligation (MSL) buffer was used because intramolecular ligation was more efficient in low salts. The composition of the MSL buffer is as follows:

5-x MSL 1-x MSL

50 mM Tris-HCl pH 7.5 10 mM Tris-HCl pH 7.5

50 mM MgCl ₂ 10 mM MgCl ₂

10 mM DTT 1 mM DTT

Analysis of in vitro ligation products was performed by PCR. When two lambda DNA restriction sequence tags of the correct size are present in the vector, an amplification product of 134 bp is formed. Smaller amplification products can be formed by inserting only one tag into an empty vector without any tag or a self-ligated vector after BsmFI decomposition of the empty vector.

Analysis of the length of the PCT product was performed using Agilent DNA500 or DNA 1000 chips. There is another method of studying in virto ligation products, in which the in vitro ligation product is transformed into competent E. coli cells, followed by analysis of plasmids isolated from individual colonies. When the first ligation product was analyzed with lambda DNA as a vector (FIG. 9B), various peaks were observed as a result of inserting DNA fragments of different lengths into the vector.

In analyzing the second ligation product, fragments of the expected size were present with the smaller fragments (FIG. 9C).

Although the first generation vector allows for size normalization with two restriction sequence tags of the expected size of lambda genomic DNA, some unexpected raw organisms have been detected. The reason is probably the self-ligation of the vector during the first ligation reaction. This may occur as a result of incomplete dephosphorylation or may be induced by DNA polymerase I treatment, which may remove the dephosphorylated base from the vector terminus. Such problems can be overcome by partial filing of genomic DNA fragments with a single restriction sequence tag as described in the Examples. For example, the BamHI site can be partially compiled with dGTP.

Alternatively, the vector can be designed by replacing the BamHI site with BglII. Ligation of the BamHI genomic fragment with a BglII digestion vector in the presence of BglII restriction enzymes will prevent self-ligation of the vector. Only expected vector-insertion ligation products will inhibit the BglII site and thus will be resistant to degradation.

BsmFI enzymes were evaluated as simple constructs. The cyclic plasmid containing 2000 bp DNA insert at the BamHI site of the first generation vector was digested with BsmFI (no site in the insert), and the 3000 bp band of the vector containing the attached DNA tag was removed from the agarose gel. Separated. These DNAs were treated with Klenow enzyme + dNTPs to generate blunt ends and treated with T4 ligase for secondary ligation. The results shown in FIG. 9 show bands of fragments smaller than the expected size of 133 bp. Did. Fragment bands of larger size appear to be PCR products because they did not appear in subsequent experiments.

These experiments indicate that the BsmFI enzyme can be correctly cleaved at the correct distance and the product of the tag can be formed successfully. Another method besides the generation of blunt ends that allows the ligation of two restriction sequence tags is to insert a linker between the two restriction sequence tags.

Another way is to invert the vector-insert-linker system. The first ligation associates genomic DNA fragments with linkers (which contain specific cleavage sites that will be useful for linearizing DNA colonies to allow sequencing of both strands of DNA amplified in each colony). After digestion with type IIS enzyme, the "vector" cancer is ligated with the cleaved end with type IIS enzyme.

2) second generation vector

In order to use two different enzymes for cloning, a second generation vector was designed that makes it possible to further reduce the average size of genomic DNA fragments, for example, and does not self-ligation of the empty vectors. To facilitate the separation of the plasmid sufficiently separated on the agarose gel from the partially digested, 1000 bp DNA fragments (from BlueScript plasmid pBSK) were included between the restriction sites of the raw vector. There is no need for dephosphorylation and treatment of DNA polymerase I for second generation vectors.

The live vector contains the insert shown in FIG. 10A, which makes it possible to use SphI and AccI restriction sites for cloning. The autologous SphI and AccI sites of the pUC19 plasmid were removed. Due to the 3′-protruding end formed by SphI digestion, the empty vector cannot self-ligation unless the Klenow enzyme completely removes its overhang. DNA digested by two different enzymes can be inserted into a second generation vector. 10A shows several possibilities for cloning.

In vitro ligation of lambda DNA digested with MspI and SphI was performed to generate SphI-AccI open vectors. Analysis of the secondary ligation product revealed a single band of the correct size as shown in FIG. 10B. Secondary ligation products were transformed into E. coli cells. Thirty colonies were inoculated with liquid culture. Twelve plasmids from the bacterial cultures with the highest densities were analyzed. No plasmid corresponding to the empty vector was observed. No plasmids with two or more base variations of the insert were observed at all. Similar experiments were performed with AluI and SphI digestion lambdas inserted into HincII and SphI digestion vectors (Alu and HincII produce blunt ends). 10C shows the analysis results of the primary ligation product. As expected, fragments of different sizes of lambda DNA were observed using Agilent 2100 bioanalyzer DNA 1000 chip. The highest peak is the size marker. 10D shows the analysis results of the product of the secondary ligation. Analysis using an Agilent 2100 bioanalyzer DNA 1000 chip revealed only a single fragment with the expected size.

6.2. Example 2: Preparation of DNA Colony Templates: Double Restriction Sequence Tag

This example illustrates the preparation of a DNA colony template containing a single restriction sequence tag from the DNA sample to be genotyped as shown in FIG. 4A. Because the restriction sequence tag, a mutable fragment, appears to be 6% smaller than the size of the DNA colony template, the size normalization step in this protocol ensures efficient and comparable amplification of all DNA colonies of all DNA colonies. Insertion into a DNA colony vector allows for the addition of a universal sequence to generate a DNA colony template.

The overall strategy of in vitro cloning used in this example is shown in Figures 11a-b. Briefly, the short double strand adapter ("short arm") consists of an amplification primer Px followed by hexanucleotide TCCGAC forming the recognition site of type IIS restriction enzyme MmeI. The 5 'end of the oligonucleotide contains a biotin moiety bound via separable disulfide bonds. The complementary strand is 5'-phosphorylated and contains an extended nucleotide compatible with the sticky end of the DNA degraded by the initiation restriction enzyme. Short arms were ligated with the DNA cleaved with the corresponding endonuclease and further treated with the type IIS enzyme MmeI. This leaves 20 bp fragments of DNA attached to short cancers. The conjugate is then purified from other DNA fragments using streptavidin beads and ligated to a "long cancer" containing another amplification primer Py.

Although the cloning strategy is based on DNA cleavage by endonucleases that recognize 6 bp sequences, digesting DNA with a second frequent cleavage enzyme (4 bp recognition site, RsaI) to reduce the average DNA fragment size is essential. desirable.

The protocols for the preparation of templates from lambda phage DNA digested with HindIII and RsaI and the different formation steps are summarized below:

Iii) digestion of lambda genomic DNA

Ii) Ligation to Short Px Cancers

Iii) decomposition by MmeI

Iii) Purification of Px Cancer-Tag Conjugates

Ⅴ) Attachment of Py arm

Viii) Purification of the final DNA colony template

The protocol of each individual step used in this example is described in detail below.

분해) digestion of bacteriophage lambda genomic DNA

10 [mu] l of lambda bacteriophage DNA (New England Biolabs, 0.5 [mu] g / [mu] l) was buffered with Y + / Tango (Fermentas); 32.5 μl H ₂ O; 1.25 μl HindIII from New England Biolabs; 1.25 μl RsaI (New England Biolabs).

Incubate at 37 ° C. for 2-16 hours.

This results in a 100 ng / μl solution of lambda phage DNA containing 42 fmol / μl of HindIII end.

HindIII protrusions are partially supplemented with dATP.

Different protocols can be used to inhibit the ligation of lambda genomic fragments and the ligation of short cancers while maximizing ligation of the lambda genomic DNA HindIII ends with short cancers.

It has been found that the best way to inhibit self-ligation at the HindIII terminus is to supplement a single base with dATP. Short cancer fragments should be designed to be compatible with the partially filled HindIII ends of genomic DNA fragments.

Supplementation of HindIII terminus:

20 μl of HidIII-RsaI digested lambda genomic DNA was added to 2 μl of 10 mM dATP; Mix with 1 μl Klenow enzyme (New England Biolabs, 5 u / μl).

Incubate at 25 ° C. for 30 minutes and 70 ° C. for 20 minutes.

Ii) ligation to short arm moieties

Care must be taken to prevent the formation of short arm dimers (or to remove them from solution) during the ligation reaction. Such dimers formed after partial MmeI degradation can result in molds of the correct size containing cloned fragments of short arms.

As mentioned above, it is a preferred method to use short cancers containing non-palindromic protrusions complementary to partially filled DNA ends. Alternatively, short arms containing dideoxy base at the 3 'end may be used. If the nick is just behind the recognition site, MmeI can cleave the DNA. The use of non-phosphorylated short cancers is another option.

This cloning step is performed using 10-fold excess moles of cancer shorter than HindIII filled with dATP.

Preparation of Short Arms:

10 μl of a 10 mM solution of biotinylated oligo short A 5′-GAGGAAAGGGAAGGGAAAGGAAGGTCCGAC-3 ′ (SEQ ID NO: 9) in Tris-HCl pH 8.0 was added to oligo short-B 5′-GCTGTCGGACCTTCCTTTCCCTTCCCTTTCCTC-3 ′ in Tris-HCl pH 8.0. Mix with 10 μl of 10 μM solution of SEQ ID NO: 10). Oligo Short-A contains cleavable disulfide bonds between biotin and the 5'-terminus.

Warm to 80 ° C. and slowly cool to room temperature for 30 minutes.

Ligation:

3 μl of 10 mM ribo ATP at the partially filled HindIII terminus of the genomic DNA mixture; 4 μl 5 μM short cancer; T4 DNA ligase (New England Biolabs, 400 u / μl) was incubated at 16 ° C. for 1 hour.

Warm to 0 ° C. and slowly cool to room temperature for 30 minutes.

Ligation:

3 μl of 10 mM riboATP at the HindIII terminus of the partially supplemented mixture of genomic DNA; 4 μl 5 μM short cancer; 1 μl of T4 DNA ligase (New England Biolabs, 400 u / μl) was added and incubated at 16 ° C. for 1 hour.

Perform DNA purification according to the Qiagen MiniElute Reaction Clean Up protocol. Elute with 12 μl of buffer EB and repeat elution with 5 μl of fresh buffer EB without changing tubes.

Under these ligation conditions, ligation of the blunt RsaI-producing ends does not result in significant polymerisation of the genomic DNA fragments.

To remove a large number of non-ligated arms, it is desirable to purify the sample using a Qiagen Mini Elute column instead of heat inactivation of T4 ligase. Double elution can increase the recovery of the reaction product.

Iii) MmeI decomposition

Effective degradation by MmiI is a critical step in determining the yield of the template. Enzymes should be used at a rate no greater than 1-2 units per μg DNA. Excess enzymes block endonuclease cleavage, according to New England Biolabs.

2 μl of buffer Y + / Tango (Fermentas) in the sample mixture; 2 μl 1 mM SAM; Add 1 μl (2 u.) MmeI (New England Biolabs).

Incubate at 37 ° C. for 1 hour.

Iii) binding / separation to streptavidin beads

According to manufacturer-supplied information, 30 minutes of time is sufficient for DNA binding with beads, but incubated overnight to increase product yield. Disulfide bond cleavage and secretion of DNA by 200 mM DTT is completed after 30 minutes. After this step, it is useful to analyze the yield of the desired product and the efficiency of MmeI degradation. Undigested product was seen as a huge DNA fragment.

Binding / secretion to SA beads:

10 μl of washed SA 280 beads (Dynal) are added to 20 μl of 2 × B & W buffer (prepared according to the Dynal protocol).

Shake overnight at room temperature. Beads are washed twice with 40 μl of 1 × B & W buffer. Beads are washed twice with 40 μl 100 mM Tris-Hcl pH 8.0. Beads are added 11 μl of 200 mM DTT in 80 mM Tris-HCl pH 8.0.

Shake for 30 minutes at room temperature.

The supernatant is separated from the beads and discarded. If necessary, 1 μl of supernatant is analyzed with an Agilent 2100 bioanalyzer DNA 1000 chip.

v) ligation of long arm moieties

This ligation is based on the recognition of two random bases present in the 3'-protrusion generated in genomic DNA by MmeI ligation. When these two bases are produced, such ligation exhibits a slow reaction and requires an increase in enzyme concentration (New England Biolabs, MmeI information note).

Manufacture of long cancer

19 μl of H 2 O in a tube with prepared PCR beads (Amersham); 1 μl of pUC19 plasmid DNA (sites 571-870 will be amplified) 1 ng / μl; 10 μΜ oligo long-A 5′-CTCACATTAA TTGCGTTGCG NNCACTGCCC GCTTTCCAG-3 ′ (SEQ ID NO: 11); Add 10 μM oligo long-B 5′-CACCAACCCAAACCAACCCAAACCGAAAAA CGCCAGCAAC G-3 ′ (SEQ ID NO: 12). Amplification is performed using a program of PTC-200 heat exchanger (MJ Research); 94 ° C. for 20 minutes 30 seconds; 25 cycles (94 ° C. 30 sec; 55 ° C. 30 sec; 72 ° C. 30 sec); Then run at 72 ° C. for 10 minutes.

The expected length of the amplification product is 323 bp. The reaction product is purified via Qiagen column and its purity and concentration is assessed with Agilent 2100 bioanalyzer DNA 1000 chip.

The PCT product must then be broken down into BtsI. The amount of enzyme and the backtime depends on the amount of PCR product. Degradation efficiency should be assessed by analysis with an Agilent 2100 bioanalyzer DNA 1000 chip. A change in size from 323 bp to 301 bp is expected. Once the digestion is complete, purification via a Qiagen column (PCR product purification protocol) is sufficient to remove small 22 bp product from the reaction. Alternatively, the 301 bp fragment should be purified via 2% agarose gel.

Ligation of long arm moieties:

2 μl of 100 mM Mgcl2 in the supernatant secreted from the beads; 2 μl 10 mM rATP; 5 μL long cancer; Add 1 μl of concentrated T4 DNA ligase (New England Biolabs, 2000 u / μl).

Incubate overnight at 16 ° C.

If necessary, 1 μl of the reaction solution is analyzed on an Agilent 2100 bioanalyzer DNA 100 chip.

Iii) final mold refining

For the final purification step, preferred ligated templates are separated from free long cancer and accidental long cancer dimers or unreacted 50 bp products. Heating of the mold under denaturing conditions should be avoided to minimize separation of the mold strands.

Final purification of the mold:

The entire sample is loaded onto a 2% agarose gel. Run until good separation between free long arm (301 bp) template (350 bp) and long arm dimer (600 bp) is achieved. The band is cut from the agarose gel.

DNA is purified by Clontech Montage Agarose Kit or Qiagen MiniElute Agarose Extraction Kit. When using the Qiagen Kit, as recommended, it will dissolve in 15 minutes at room temperature without the tube warming to 50 ° C. If necessary, the final product can be analyzed on an Agilent 2100 bioanalyzer DNA 1000 chip.

Then size standardization was demonstrated. The same experiment was performed in parallel from bacteriophage lambda genomic DNA or human genomic DNA labeled with ³³ P-dATP.

11C shows the results of radiographic analysis of the aliquots collected after the various steps of the process. Lane 1: PCR product of 350 bp of complete DNA colony vector size; Lanes 2-6: lambda genomic DNA and lanes 7-10 human genomic DNA; Lanes 3 and 7 after ligation into short arms; Lanes 4 and 8, size leveling after degradation with MmeI; Lanes 5, 6, 9, and 10: DNA colony vectors were generated at desired sizes after ligation into long cancers.

DNA colonies were then generated as follows: WO 00 / using DNA colony vectors containing lambda or human genomic DNA fragments digested with HindIII and size-equalized with MmeI, prepared as directed in this example. DNA colonies were generated by the method of 18957. 11D shows DNA colonies of lambda DNA. 11E shows lambda DNA (left column) or human DNA (first three images of right column). This DNA colony is then sequenced in situ using the method of WO 98/44152 to identify restriction sequence tags.

The size of the DNA colony vector was also verified by PCR amplification. PCR products are then cloned into pUC19 plasmid and transformed in E. coli campentant cells (XL-2 Blue, Stratagene). Minipreps from individual clones were sequenced. It was demonstrated that the restriction sequence tag was 20bp, the expected size. However, 21 base long tags were recovered in some clones. No tag with less than 20 bases was found at all.

Fingerprinting experiments demonstrated that all expected 14 HindIII-digested lambdas were present in the DNA colony vector. After MmeI treatment and long cancer ligation, fragments were purified from agarose gels and primer extension was performed in the presence of 3 dXTP and one dideoxy nucleotide (eg dATP, dTTP, dCTP, and ddGTP). The product was then analyzed on acrylamide gels to enable identification of each expected fragment.

When using a 6 base cutter for cloning, which produces 4 protrusions, information on 21 consecutive bases can be obtained from the already prepared template. Of the 21, 6 are known bases that form the recognition site of endonucleases and 15 can be used to detect gene mutations. For some enzymes, if the "sticky" end of a short cancer overlaps the MmeI site, this number can be increased, for example, if TCCGA ligated to the NcoI end CATGG forms the MmeI site.

In addition, two variants of the standard protocol are used to increase the power of cloning. In one variation, flat end-generating enzymes were used. If the enzyme used to cleave the DNA has six base recognition sequences and leaves the blunt ends, information on 23 consecutive bases (6 known and 17 SNP detected) can be obtained. Since the efficiency of ligation at the smooth end is lower, an extension of the ligation time is necessary. Nevertheless, if ligation is done overnight, sufficient ligation efficiency can be obtained. The efficiency of the template obtained using MscI-digested lambda DNA was similar to the yield of HindIII digested lambda DNA.

Analysis of the plasmid obtained by the insertion of the amplified template into the pUC19 plasmid revealed the following:

(1) the absence of "moulds" containing short cancer dimers;

(2) small amounts of undesirable product (only 1-2 of 18 clones);

(3) Excellent emergence of different lambda genomic DNA fragments of the template (only three fragments of 15 templates were found twice).

In another variation, artificial production of blunt ends was used. Upon removal of the 4 base overhangs remaining after the initial DNA digestion, the cloning information increases to 25 bases (6 known and 19 SNP detected). In preliminary experiments, two enzymes were examined that could eliminate protrusions. Mung bean nucleases (New England Biolabs) failed to efficiently produce smooth ends. Removal of the 3 'overhang by Klenow enzyme yielded satisfactory results. In addition, in the presence of the essential deoxynucleotides, the latter enzyme can also inhibit the terminal produced by frequent cutters from participating in ligation. For example, if DNA is digested with PstI and MspI, the Klenow enzyme will trim the PstI terminus in the presence of dCTP and convert the MspI terminus into an inactive single base 5 ′ overhang (FIG. 12).

6.3. Example 3: Detection of Genome-Wide Sequence Variations

This example describes an embodiment of the invention used for the generation of a large number of restriction sequence tags from a complex genome in a reproducible manner. This restriction sequence tag is useful for identifying genetic variations between genomes without prior knowledge of mutations, and in a comprehensive way is useful for identifying variants related to phenotypes specific to a population of individuals without hypotheses based on prior knowledge, and obtained restriction sequences. Due to their high density, they are useful for associating such variations with at least genomic regions of size.

The method disclosed in this example is based on the use of the same restriction endonuclease to generate the same restriction fragment from different genomic DNA samples. After amplification, the ends of these restriction fragments are sequenced and the sequences are processed to identify restriction sequence tags, which are short sequences of nucleotides present immediately next to the recognition sites of restriction enzymes used for digestion of genomic DNA.

For each patient in the population under study, a patient under clinical study, follow these steps:

1) Extraction of Genomic DNA

Genomic DNA is extracted from biological samples by different individuals. Such a biological sample is a swab or blood sample of the ball. Genomic DNA is extracted using standard protocols. Typically, 0.5-3 μg of genomic DNA is extracted from a ball swab and 4 μg of genomic DNA is extracted from 100 μl of whole blood sample. Since one double fold human genome has about 6 pg of DNA, this corresponds to more than 80 to 600 copies of the double fold genome, which is sufficient for our purposes.

2) limited disassembly

The restriction endonuclease to be used is selected according to the density of the restriction sequence tag directly dependent on the average distance between two restriction enzyme recognition sites (equivalent to the average distance of the genomic restriction fragment to be obtained). Therefore, since the goal is to obtain at least one cleavage on average every 5000 bases, a restriction enzyme with 6 base recognition sites is used, which is expected to produce fragments of average size of 4096 bases. Thus, 1,400,000 genomic restriction fragments of each two-fold human genome with about 6 billion bases are generated. For each genomic restriction fragment, because two restriction sequence tags are formed, the total number of more than 2.8 million different restriction sequence tags is generated in the two fold human genome. As discussed below, the restriction sequence tag generated in this example has 15 bases in length, polymorphisms are found every 500 bases in the human genome, and 2.8 million tags generate 80,000 polymorphs per patient, or It is estimated that one polymorph is produced for every 35,000 bases of the genomic sequence.

The number of restriction sequence tags obtained per individual can be controlled by using different restriction enzymes or restriction enzyme combinations. For example, to increase the number of restriction sequence tags, multiple restriction enzymes can be used in combination or the method can be repeated in sequence with different enzymes. Alternatively, enzymes with longer recognition sites can be used alone or in combination to reduce the number of restriction sequence tags.

When the method is repeated in the same sample or in different samples, it is essential to generate the same restriction fragments except for variations due to changes in the genomic sequence between the samples, thus obtaining the same restriction sequence tag. In theory, different restriction enzymes with the same recognition site may be used, such as isoschizomers. However, this example uses the same enzymes from the same origin and from the same supplier.

Restriction digestion is performed using 10-20 copies of the 2-fold genome per patient, and repetitions are performed to identify each restriction sequence tag.

3) Insertion of genome restriction fragments into amplification and sequencing vectors

In this example, DNA colonies are used for amplification and sequencing of genomic restriction fragments.

The genome restriction fragment is linked to a DNA colony vector, ie an engineered nucleic acid having a predetermined sequence, by performing a ligation reaction to generate a cyclic molecule. The DNA colony vector contains the following features: It has two ends that are compatible and preferably tacky with the ends of the digested genomic DNA fragments, the ends being dephosphorylated to inhibit self ligation of the vector; Having two recognition sites for type IIS such as BsmFI, BceAI, Eco57I, or MmeI, each recognition site is oriented to induce cleavage in genomic restriction fragments located at the ends or linked to the vector; Having recognition sites for two sequencing primers, each of which is oriented such that primer extension is possible in the direction of genomic restriction fragments that are located close to or linked to the ends of the vector; Having two amplification primers oriented to enable amplification of the inserted fragments and portions of the vector, which may overlap with the sequence of the sequencing primers; And optionally, a recognition site for a rare cleavage restriction enzyme located outside the site to be amplified using the amplification primer sequence. Additional features of DNA colony vectors include additional restriction sites or spacer sequences, for example, at sites to be amplified for linearization of DNA colonies.

To inhibit concatemerization of genomic restriction fragments, DNA colony vector molecules are used in excess moles relative to genome restriction fragments.

4) Standardization of insert size

The cyclic DNA molecule containing the DNA colony vector linked to the genome restriction fragment is then digested with type-IIS restriction enzyme. For example, using BceAI will cleave 14 bases in the inserted genomic fragment. After a reaction supplemented with DNA polymerase, such as the Klenow fragment of DNA polymerase I or T4 DNA polymerase, the resulting smooth ends are ligated to assemble 28 base sites of each of the 28 genomic restriction fragments, ie, genomic restriction fragments. From the ends yield cyclic molecules containing 14 base moieties.

Longer inserts are generated using enzymes such as MmeI that cleave 20 bases outside the recognition site. However, due to the fact that 2-base 3 'overhangs are produced, the reaction with DNA polymerase, such as the Klenow fragment of DNA polymerase I or T4 DNA polymerase, will remove two bases. In this case, the resulting genomic restriction fragment is 36 bases in length.

5) Generation of DNA Colony Templates

DNA colony templates are generated using one or more PCR amplification cycles in the presence of amplification primers. The DNA template molecular sequence comprises the following sequence from the 5 'end to the 3' end: the first amplification primer sequence in the forward direction; The sequence of the forward first sequencing primer (which may overlap with the sequence of the first amplification primer); a first recognition site of type-IIS restriction enzyme; 28 or 36 bases linked to genomic restriction fragments resulting from the size normalization step (including half the recognition sites of restriction enzymes used to degrade genomic DNA); second recognition site of type-IIS restriction enzyme; The sequence of the reverse sequencing primer (which may overlap with the sequence of the second amplification primer sequence); And a sequence of reverse a second amplification primers.

Alternatively, the DNA colony template can be generated by simple restriction digestion using a rare cleavage enzyme that cleaves the DNA colony vector outside the site amplified by the amplification primers.

6) Generation of DNA Colonies

The first step to generate DNA colonies is to attach the DNA colony template molecules and amplification primers onto a solid surface, such as a functionalized glass or a plastic surface such as NucieoLink tubes (Nunc, Roskide, DK). The concentration of the DNA colony template and amplification primer molecules is chosen so that the surface is covered with a dense amplification primer molecule after attachment, and the DNA colony template molecule is subjected to local amplification with DNA colonies using an amplification primer to which the DNA colony template molecule is attached. Make it possible to happen and choose the appropriate repetition. In an example where 1.4 million restriction fragments are generated, about 30 million DNA colonies are generated per surface cubic centimeter.

Amplification is performed using an isothermal method (as described in Section 5.3 and PCT Publication WO 02/46456).

7) Sequencing DNA Colonies

After amplification, DNA colonies become single stranded by denaturation after restriction digestion. The first sequencing primer is then hybridized to the DNA colony vector. The surface is then incubated with a mixture of only one of four possible nucleotides and a DNA polymerase such as T7 DNA polymerase. The mixture may be labeled with fluorescence or may be of the same kind that is not labeled, allowing about one of the 10 integrated nucleotides to be labeled with fluorescence. Such labeled nucleotides integrate into the 3 'end of the primer if they are complementary to the sequence of the DNA colony molecule. After the primer extension step, images are taken with a fluorescence microscope (Axiovert 200, Zeiss, Germany equiped with ORCA-ER CCD camera, Hamamatsu, Japan) to determine the intensity and location of the fluorescence of each DNA colony. This process is repeated in a stepwise manner by periodically repeating four different kinds of nucleotides one by one. In each step, a given base is used for integration, and the resulting signal is then measured for each DNA colony on the surface. In each step, the fluorescence intensity of DNA colonies incorporating one or more bases is increased proportionally, while the fluorescence intensities of colonies not incorporating bases remain unchanged. By comparing the fluorescence intensity after the integration step with the fluorescence intensity before it, the amount of base incorporated into the DNA colony is determined. The sequence of DNA contained in each DNA colony is determined by tracking the sequential change in fluorescence intensity in each DNA colony and linking the intensity with the identity of the base used in the extension step.

The sequencing step is repeated until 28 or 36 bases are read from the genomic fragment. The number of bases that are sequenced can be reduced by using sequencing primers that extend to half of the recognition site of the restriction enzyme used for digestion of genomic DNA.

If necessary, the extended first sequencing primer can be removed by denaturation and washing, and sequencing of the complementary strand can be performed using a second sequencing primer.

8) restriction enzyme tag

Sequences obtained by sequencing DNA colonies are processed to identify two restriction sequence tags from each original genome restriction fragment. For example, when using the enzyme MmeI for standardization of the size of the linked restriction fragment, the restriction sequence tag is 18 bases long and 3 bases missing from half of the restriction sites used for digestion of genomic DNA. to be. When using BceAI, the restriction sequence tag is 11 bases in length.

These two restriction sequence tags represent the ends of the original genomic restriction fragment. The two tags obtained from each DNA colony are physically close to the genome (eg, on average 4096 bases apart) and stored for another use. The location of the tag on the genome is determined using a sequence of six bases, ie 21 or 17 base sequences, of the restriction sites of the restriction enzyme used for digestion of genomic DNA, along with 15 or 11 bases.

9) Aligning Restriction Sequence Tags and Identifying Sequence Variations Associated with Phenotype

The restriction sequence tags are then compared using a computer program to identify different tags and determine the number of restriction sequence tags for each individual. These tags are then compared among individuals to identify sequence variations associated with particular phenotypes of homologous tag groups and populations. Comparisons can be made by statistical analysis known in the art, such as hidden Markov chains or clustering methods. Tags can also be compared with sequences from tags or databases that have already been obtained.

Comparison of restriction sequence tags between two populations can produce different results. For a given sequence variation, the proportion of the two types of genetic variation in population 1 may differ from that in population 2.

In other cases, the proportion of various types of sequence variation may be similar or identical in the two populations, but analysis of specific combinations of different genetic variations in an individual may indicate that a combination of variations is expressed in different proportions in the two populations. I can tell you.

Examples of tag groups that can be obtained in the method of the present invention are as follows:

In entity 1 it is determined as follows:

.

In entity 2, it is determined as follows:

In entity 3 it is determined as follows:

From the above results,

The tags T1a, T2a, and T3a are identical and form a group g1 of the group-sequence Sg1 = T1a

The tags T1b and T2b are identical and form a group-sequence group g2 Sg2 = T2b

The tags T1c and T3b are identical and form a group-sequence group g3 Sg3 = T1c

The tags T1e and T2c are identical and form a group-sequence group g4 Sg4 = T1e

The tags T1f and T3c are identical and form a group-sequence group g5 Sg5 = T1f.

It can be said that

Are identical except for one single base, but each of them is very different from Sg1, Sg4, and Sg5,

Are identical except for one single base, but each of them is very different from Sg1, Sg2, and Sg3.

Then, groups G1 formed by Sg2 and Sg3, groups G2 formed by Sg4 and Sg5, and group G3 formed by group Sg1 can be generated.

Because each individual has two different sets of chromosomes,

(1) Subject 1 has two Sg1 copies, one Sg2 copy, one Sg3 copy, one Sg4 copy, and one Sg5 copy,

(2) Subject 2 has two Sg1 copies, two Sg2 copies, and two Sg4 copies,

(3) Subject 3 can be considered to have two Sg1 copies, two Sg3 copies, and two Sg5 copies.

Typical results 1

In cohort 1 it was found:

Sequence tag Sg1 1000 copies

Copy of sequence tag Sg2 327

Copy of sequence tag Sg3 673

Copy of sequence tag Sg4 521

Copy of sequence tag Sg5 479

In cohort 2 it was found:

Sequence tag Sg1 1000 copies

Copy of sequence tag Sg2 345

Copy of sequence tag Sg3 665

Copy of sequence tag Sg4 502

Copy of sequence tag Sg5 498

Since there is no significant difference in the respective composition of groups G1, G2, and G3 between group 1 and group 2, it can be concluded that these groups are not related to the difference in phenotype between groups.

Typical results 2

In cohort 1 it was found:

Sequence tag Sg1 1000 copies

Copy of sequence tag Sg2 993

7 copies of the sequence tag Sg3

Copy of sequence tag Sg4 521

Copy of sequence tag Sg5 479

In cohort 2 it was found:

Sequence tag Sg1 1000 copies

Copy of sequence tag Sg2 946

54 copies of the sequence tag Sg3

Copy of sequence tag Sg4 502

Copy of sequence tag Sg5 498

Since there is no significant difference in the respective composition of groups G1 and G3 between group 1 and group 2, it can be concluded that these groups are not related to the difference in phenotype between groups. Since there is a significant difference in the composition of group G2 between group 1 and group 2, it can be concluded that this group is related to the difference in phenotype between groups. In addition, individuals with Sg3 sequences are more likely to belong to population 2 than individuals with Sg2.

Typical results 3

In cohort 1 it was found:

Sequence tag Sg1 1000 copies

Copy of sequence tag Sg2 314

Copy of sequence tag Sg3 686

Sequence tag Sg4 486 copies

Copy of sequence tag Sg5 514

In cohort 2 it was found:

Sequence tag Sg1 1000 copies

Copy of sequence tag Sg2 289

Copy of sequence tag Sg3 711

Copy of sequence tag Sg4 511

Copy of sequence tag Sg5 489

There is no significant difference in the composition of each of groups G1, G2, and G3 between group 1 and group 2. However, we can further analyze the data by counting how many combinations of sequence tags an individual has:

This analysis shows significant differences in the combination of sequence tags between population 1 and population 2. Thus, this combination of sequence tags is related to phenotypic differences between populations.

7. Cited References

All references cited herein are hereby incorporated by reference in their entirety, and each publication or patent or patent application is specifically incorporated for all purposes to the full extent and for all purposes for each purpose. Are integrated.

Many modifications and variations of the present invention can be made without departing from the spirit and scope of the invention, which is apparent to those skilled in the art. The specific embodiments described herein are provided by way of example only, and the invention is to be limited only by the appended claims and equivalents thereof as claimed in those claims.

Claims (102)

I) A) digesting the nucleic acid of each individual organism using one or more first restriction enzymes to produce a set of restriction fragments; And B) determining one or more restriction sequence tags for each of the restriction fragments, wherein the one or more restriction sequence tags comprise a sequence of corresponding restriction fragments, the set of restriction sequence tags of each individual organism Thereby generating a restriction sequence tag set of each individual organism of the one or more individual organisms; And II) classifying the restriction sequence tags of the one or more individual organisms into one or more restriction sequence tag groups comprising homologous restriction sequence tags that identify sequence variations associated with the phenotype; A method of determining genome wide sequence variation associated with a phenotype of one or more individual organisms. The method of claim 1, wherein determining the restriction sequence tag set B1) linking the restriction fragment in the set of restriction fragments with a first engineered nucleic acid comprising a predetermined nucleotide sequence to obtain a first annular nucleic acid fragment, wherein the predetermined nucleotide sequence is determined by the second restriction enzyme by the restriction enzyme. At least one recognition site of a second restriction enzyme, positioned and oriented to cleave at the fragment; B2) digesting the first cyclic nucleic acid fragment with the second restriction enzyme; B3) altering the termini produced by the second restriction enzyme to enable ligation; B4) linking the ends produced by the second restriction enzyme to generate a second set of circular nucleic acid fragments; And B5) sequencing at least a portion of said respective restriction fragment of said second circular nucleic acid to determine said set of restriction sequence tags. 3. The method of claim 2, wherein each of the one or more recognition sites is located close to the terminus of the first engineered nucleic acid. 4. The method of claim 2 or 3, wherein each of the one or more recognition sites are located less than 25 nucleotides away from the end of the first engineered nucleic acid. The method of claim 2, wherein each of the one or more recognition sites is located less than 5 nucleotides away from the end of the first engineered nucleic acid. The method of any one of claims 2 to 5, wherein the second restriction enzyme is a type IIS endonuclease. The method according to any one of claims 2 to 6, further comprising the step of immobilizing and amplifying the nucleic acid fragment contained in the second annular nucleic acid sequence on a solid surface before the step B5). . 8. The method of claim 7, wherein said fixing and amplifying step is performed by producing a colony of said nucleic acid fragment in said second circular nucleic acid fragment on said solid surface, said nucleic acid fragment colony being said nucleic acid fragment in said second circular nucleic acid fragment. And a plurality of immobilized single stranded DNA molecules comprising one of the two. The method of claim 8, Iii) linearizing the second circular nucleic acid fragment to produce a linear fragment; Ii) providing a solid surface comprising a plurality of colony primers fixed at the 5 'end on the solid surface, wherein each colony primer comprises a sequence that is hybridizable with the sequence at the 3' end of the linear fragment Step to be; Iii) denaturing the linear fragments to produce single stranded fragments; Iii) annealing the single stranded fragment to the immobilized colony primer; Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments to the fixed colony primers; Iii) repeating said steps v) to iii) such that said colonies are produced at each particular site on said solid surface. The method of claim 8, Iii) linearizing the second circular nucleic acid fragment to produce a linear fragment; Ii) mixing the linear fragment with colony primers each comprising a sequence hybridizable with a sequence at the 3 'end of the linear fragment; Iii) implanting the linear fragments and colony primers at 5 'ends on a solid surface to produce fixed linear fragments and fixed colony primers; Iii) denaturing the fixed linear fragment to produce a fixed single stranded fragment; v) annealing the fixed single stranded fragment to the fixed colony primer to obtain an annealed single stranded fragment; Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments to the fixed colony primers; Iii) repeating said steps v) to iii) such that said colonies are produced at each particular site on said solid surface. The method of claim 8, Iii) linearizing the second circular nucleic acid fragment to produce a linear fragment; Ii) mixing the linear fragments with colony primers, each of which comprises a sequence hybridizable with a sequence at the 3 'end of the linear fragment, wherein the concentration of the colony primers is adjusted to allow amplification of the implanted linear fragments to occur. step; Iii) implanting the linear fragments and colony primers at 5 'ends on a solid surface to produce fixed linear fragments and fixed colony primers; Iii) applying the amplification solution containing polymerase and nucleotides to said solid surface such that said colonies are isothermally produced at a specific location on said solid surface, wherein said colonies are produced. . The method of claim 9, wherein the sequencing is Iii) hybridizing a sequencing primer to the colony; Ii) performing primer extension with one labeled nucleotide; Iii) detecting the amount of labeled nucleotides incorporated into each of the extended primers at said location; And Iii) repeating steps ii) and iii) to determine a portion of the nucleotide sequence of each colony. 13. The method of claim 12, wherein the labeled nucleotides are nucleotides labeled with fluorescence and the detection involves detecting the fluorescence intensity of the labeled nucleotides. The method of claim 1, wherein the first restriction enzyme cleaves at both recognition sites in such a way that the cleavage site surrounds a portion of the sequence that is not part of the recognition site, and determining the restriction sequence tag set B1) altering the termini produced by the first restriction enzyme to enable ligation; B2) linking said restriction fragment in said set of restriction fragments with a first engineered nucleic acid comprising a predetermined nucleotide sequence to obtain a first set of circular nucleic acid fragments; And B3) sequencing at least a portion of said respective restriction fragment of said first circular nucleic acid to determine said set of restriction sequence tags. The method of claim 14, further comprising the step of immobilizing and amplifying the nucleic acid fragment contained in the second annular nucleic acid fragment on the solid surface before step B3). 16. The method of claim 15, wherein the fixing and amplifying step is performed by generating colonies of the nucleic acid fragments in the first annular nucleic acid fragment on the solid surface, each of the colonies being in the nucleic acid fragments in the first annular nucleic acid fragment. A method comprising a plurality of immobilized single stranded DNA molecules. The method of claim 16, Iii) linearizing the first circular nucleic acid fragment to produce a linear fragment; Ii) providing a solid surface comprising a plurality of colony primers immobilized at the 5 'end on the solid surface, wherein each colony primer comprises a sequence that is capable of hybridizing with the sequence at the 3' end of the linear fragment Step to be; Iii) denaturing the linear fragments to produce single stranded fragments; Iii) annealing the single stranded fragment to the immobilized colony primer; Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments to the fixed colony primers; Iii) repeating said steps v) to iii) such that said colonies are produced at each particular site on said solid surface. The method of claim 16, Iii) linearizing the first circular nucleic acid fragment to produce a linear fragment; Ii) mixing the linear fragment with colony primers each comprising a sequence hybridizable with a sequence at the 3 'end of the linear fragment; Iii) implanting the linear fragments and colony primers at 5 'ends on a solid surface to produce fixed linear fragments and fixed colony primers; Iii) denaturing the fixed linear fragment to produce a fixed single stranded fragment; v) annealing the fixed single stranded fragment to the fixed colony primer to obtain an annealed single stranded fragment; Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments to the fixed colony primers; Iii) repeating said steps v) to iii) such that said colonies are produced at each particular site on said solid surface. The method of claim 16, Iii) linearizing said first type nucleic acid fragment to produce a linear fragment; Ii) mixing the linear fragments with colony primers, each of which comprises a sequence hybridizable with a sequence at the 3 'end of the linear fragment, wherein the concentration of the colony primers is adjusted to allow amplification of the implanted linear fragments to occur. step; Iii) implanting the linear fragments and colony primers at 5 'ends on a solid surface to produce fixed linear fragments and fixed colony primers; Iii) applying the amplification solution containing polymerase and nucleotides to said solid surface such that said colonies are isothermally produced at a specific location on said solid surface, wherein said colonies are produced. . The method of claim 17, wherein the sequencing is Iii) hybridizing a sequencing primer to the colony; Ii) performing primer extension with one labeled nucleotide; Iii) detecting the amount of labeled nucleotides incorporated into each of the extended primers at said location; And Iii) repeating steps ii) and iii) to determine a portion of the nucleotide sequence of each colony. 21. The method of claim 20, wherein the labeled nucleotides are nucleotides labeled with fluorescence and the detection involves detecting fluorescence intensity of the labeled nucleotides. The method of claim 1, wherein determining the restriction sequence tag set B1) a first engineered nucleic acid comprising a predetermined nucleotide sequence comprising a recognition site of a second restriction enzyme positioned and oriented so that the second restriction enzyme in the restriction fragment is cleaved and the restriction fragment in the set of restriction fragments Linking to obtain a first set of nucleic acid fragments; B2) digesting the first nucleic acid fragment with the second restriction enzyme; B3) altering the termini produced by the second restriction enzyme to enable ligation; B4) linking said end produced by said second restriction enzyme with a second engineered nucleic acid comprising a predetermined nucleotide sequence to generate a second nucleic acid fragment; And B5) sequencing at least a portion of each respective restriction fragment of the second nucleic acid fragment to determine the set of restriction sequence tags. 23. The method of claim 22, wherein said recognition site of said second restriction enzyme is located close to the terminus of said first engineered nucleic acid. 24. The method of claim 22 or 23, wherein each of the one or more recognition sites are located less than 25 nucleotides away from the end of the first engineered nucleic acid. 25. The method of any one of claims 22 to 24, wherein the recognition site is located 0 to 5 nucleotides away from the end of the first engineered nucleic acid. 26. The method of any one of claims 22 to 25, wherein the second restriction enzyme is a type IIS endonuclease. 27. The method of any one of claims 22 to 26, further comprising the step of immobilizing and amplifying the nucleic acid fragment in the second nucleic acid fragment on a solid surface before the step B5). 28. The method of claim 27, wherein said fixing and amplifying step is performed by producing a colony of said nucleic acid fragment in said second nucleic acid fragment on said solid surface, said nucleic acid fragment colony being in said nucleic acid fragment in said second nucleic acid fragment. A method comprising a plurality of immobilized single stranded DNA molecules. The method of claim 28, i) each colony primer comprises a sequence that is hybridizable with a sequence at the 3 'end of the second nucleic acid fragment and provides a solid surface comprising a plurality of colony primers immobilized at the 5' end on the solid surface. step; Ii) denaturing said second nucleic acid fragment to produce a single stranded fragment; Iii) annealing the single stranded fragment to the immobilized colony primer; Iii) generating an immobilized double stranded nucleic acid fragment by performing a primer extension reaction using the annealed single stranded fragment as a template; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments with fixed colony primers; Iii) repeating said steps iii) to iii) such that said colonies are formed at each particular location of said solid surface. The method of claim 28, Iii) mixing the second window nucleic acid fragment with colony primers, each of which comprises a sequence hybridizable with a sequence at the 3 'end of the linear fragment; Ii) implanting said second nucleic acid fragment and colony primer to the 5 'end on a solid surface to produce immobilized nucleic acid fragment and immobilized colony primer; Iii) denaturing the immobilized nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragment to the fixed colony primer to obtain an annealed single stranded fragment; Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments to the fixed colony primers; Iii) repeating said steps v) to iii) such that said colonies are produced at each particular site on said solid surface. The method of claim 28, Iii) mixing the second nucleic acid fragment with a colony primer each comprising a sequence capable of hybridizing with a sequence at the 3 'end of the second nucleic acid fragment, wherein the concentration of the colony primer is amplified of the second nucleic acid fragment to be implanted Adjusting to make this happen; Iii) grafting said second nucleic acid fragment and colony primer to the 5 'end on a solid surface to produce immobilized linear fragments and immobilized colony primers; Iii) applying the amplification solution containing polymerase and nucleotides to said solid surface such that said colonies are isothermally produced at a specific location on said solid surface, wherein said colonies are produced. . 32. The method of any of claims 29-31, wherein the sequencing is Iii) hybridizing a sequencing primer to the colony; Ii) performing primer extension with one labeled nucleotide; Iii) detecting the amount of labeled nucleotides incorporated into each of the extended primers at said location; And Iii) repeating steps ii) and iii) to determine a portion of the sequence of each colony. 33. The method of claim 32, wherein the labeled nucleotides are fluorescently labeled nucleotides, and wherein the detection involves detecting fluorescence intensity of the labeled nucleotides. The method of claim 1, wherein the first restriction enzyme is a rare cutter, and the determining of the restriction sequence tag set comprises: B1) linking said restriction fragment in said restriction fragment set with a first engineered nucleic acid comprising a predetermined nucleotide sequence to obtain a first set of nucleic acid fragments; B2) digesting the first nucleic acid fragment with one or more second restriction enzymes that differ from the first restriction enzyme and do not cleave the first engineered nucleic acid to obtain a second restriction fragment; B3) linking the end of the second restriction fragment with a second engineered nucleic acid comprising a predetermined nucleotide sequence to form a second set of nucleic acid fragments; B4) sequencing at least a portion of said respective restriction fragment of said second nucleic acid fragment to determine said restriction sequence tag set. 35. The method of claim 34, wherein the rare cutter recognizes a six-base recognition sequence. 35. The method of claim 34, wherein the rare cutter recognizes an 8-base recognition sequence or at least 8 base recognition sequences. 37. The method of any one of claims 34 to 36, further comprising the step of immobilizing and amplifying the nucleic acid fragment of the second nucleic acid fragment on a solid surface before step B4). 38. The colony of the nucleic acid fragment of the second nucleic acid fragment as recited in claim 37 wherein said fixing and amplifying comprises a plurality of immobilized single-stranded DNA molecules of one of said nucleic acid fragments of said second nucleic acid fragment. By producing on a solid surface. The method of claim 38, Iii) providing a solid surface comprising a plurality of colony primers, wherein a colony primer comprising a sequence hybridizable with a sequence at the 3 'end of said second nucleic acid fragment is immobilized at the 5' end on a solid surface; Ii) denaturing said second nucleic acid fragment to produce a single stranded fragment; Iii) annealing the single stranded fragment to the immobilized colony primer; Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments to the fixed colony primers; Iii) repeating said steps iii) to iii) to produce said colonies at each particular site on said solid surface. The method of claim 38, Iii) mixing the second nucleic acid fragment with colony primers each comprising a sequence capable of hybridizing with a sequence at the 3 'end of the second nucleic acid fragment; Ii) implanting said second nucleic acid fragment and colony primer to the 5 'end on a solid surface to produce an immobilized second nucleic acid fragment and immobilized colony primer; Iii) denaturing the fixed linear fragment to produce a fixed single stranded fragment; Iii) annealing the fixed single stranded fragment to the fixed colony primer to obtain an annealed single stranded fragment; Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments to the fixed colony primers; Iii) repeating said steps iii) to iii) to produce said colonies at each particular site on said solid surface. The method of claim 38, Iii) mixing the second nucleic acid fragment with a colony primer each comprising a sequence capable of hybridizing with a sequence at the 3 'end of the second nucleic acid fragment, wherein the concentration of the colony primer is Adjusted to allow amplification to occur; Iii) implanting said second nucleic acid fragment and colony primer to the 5 'end on a solid surface to produce an immobilized second nucleic acid fragment and immobilized colony primer; Iii) applying the amplification solution containing polymerase and nucleotides to said solid surface such that said colonies are isothermally produced at a specific location on said solid surface, wherein said colonies are produced. . 42. The method of any one of claims 39 to 41 wherein said sequencing is Iii) hybridizing a sequencing primer to the colony; Ii) performing primer extension with one labeled nucleotide; Iii) detecting the amount of labeled nucleotides incorporated into each of the extended primers at said location; And Iii) repeating steps ii) and iii) to determine a portion of the nucleotide sequence of each colony. 43. The method of claim 42, wherein the labeled nucleotides are nucleotides labeled with fluorescence and the detection involves detecting the fluorescence intensity of the labeled nucleotides. The method of claim 1, wherein determining the restriction sequence tag set B1) linking said restriction fragment in said set of restriction fragments with a first engineered nucleic acid comprising a predetermined nucleotide sequence to obtain a first set of nucleic acid fragments; And B2) obtaining a second restriction fragment by digesting the first nucleic acid fragment with a second restriction enzyme that is different from the first restriction enzyme and that does not cleave the first nucleic acid; B3) linking the end of the second restriction fragment with a second engineered nucleic acid comprising a predetermined nucleotide sequence to generate a second set of nucleic acid fragments; B4) sequencing at least a portion of each said nucleic acid fragment in said second nucleic acid fragment to determine said set of restriction sequence tags. 45. The method of claim 44, further comprising repeating steps B2) through B4) for a plurality of different second restriction enzymes. 46. The method of claim 45, further comprising the step of immobilizing and amplifying the nucleic acid fragments in said second nucleic acid fragments on a solid surface prior to said step B4). 47. The method of claim 46, wherein the fixing and amplifying step is performed by generating colonies of the nucleic acid fragments in the second nucleic acid fragment on the solid surface, each of the colonies being one of the nucleic acid fragments in the second nucleic acid fragment. A method comprising a plurality of immobilized single stranded DNA molecules. The method of claim 47, i) providing a solid surface comprising a plurality of colony primers, wherein each colony primer comprises a sequence hybridizable with a sequence at the 3 'end of the second nucleic acid fragment and is immobilized at the 5' end on the solid surface. ; Ii) denaturing said second nucleic acid fragment to produce a single stranded fragment; Iii) annealing the single stranded fragment to the immobilized colony primer; Iii) generating an immobilized double stranded nucleic acid fragment by performing a primer extension reaction using the annealed single stranded fragment as a template; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments with fixed colony primers; Iii) repeating said steps iii) to iii) such that said colonies are formed at each particular location of said solid surface. The method of claim 47, Iii) mixing the second nucleic acid fragment with colony primers each comprising a sequence capable of hybridizing with a sequence at the 3 'end of the second nucleic acid fragment; Ii) implanting said second nucleic acid fragment and colony primer to the 5 'end on a solid surface to produce an immobilized second nucleic acid fragment and immobilized colony primer; Iii) denaturing the immobilized second nucleic acid fragment to produce an immobilized single stranded fragment; Iii) annealing the fixed single stranded fragment to the fixed colony primer to obtain an annealed single stranded fragment; Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments to the fixed colony primers; And Iii) repeating said steps iii) to iii) to produce said colonies at each particular site on said solid surface. The method of claim 47, Iii) mixing the second nucleic acid fragment with a colony primer each comprising a sequence capable of hybridizing with a sequence at the 3 'end of the second nucleic acid fragment, wherein the concentration of the colony primer is amplified of the second nucleic acid fragment to be implanted Adjusting for this to occur; Iii) implanting said second nucleic acid fragment and colony primer to the 5 'end on a solid surface to produce fixed linear fragments and fixed colony primers; Iii) applying the amplification solution containing polymerase and nucleotides to said solid surface such that said colonies are isothermally produced at a specific location on said solid surface, wherein said colonies are produced. . 51. The method of any of claims 48-50, wherein the sequencing is Iii) hybridizing a sequencing primer to the colony; Ii) performing primer extension with one labeled nucleotide; Iii) detecting the amount of labeled nucleotides incorporated into each of the extended primers at said location; And Iii) repeating steps ii) and iii) to determine a portion of the sequence of each colony. 53. The method of claim 51, wherein said labeled nucleotides are nucleotides labeled with fluorescence and said detection involves detecting the fluorescence intensity of said labeled nucleotides. The method of claim 1, wherein determining the restriction sequence tag set B1) the restriction fragment in the restriction fragment set, wherein the second restriction enzyme and the third restriction enzyme include two recognition sites of the second restriction enzyme and two restriction sites of different restriction enzymes; A recognition site is located between two recognition sites of the third restriction enzyme and the recognition site of the third restriction enzyme comprises a first nucleotide sequence comprising a predetermined nucleotide sequence located and oriented so that the third restriction enzyme is cleaved at the restriction fragment. Linking with the engineered nucleic acid of to obtain a first set of circular nucleic acid fragments; B2) digesting the first nucleic acid fragment with a second restriction enzyme to obtain a second nucleic acid fragment; B3) joining the ends of said second restriction fragment to produce a second set of circular nucleic acid fragments; B4) sequencing a portion of each of said restriction fragments in said third circular nucleic acid fragment to determine said restriction sequence tag set. The method of claim 53, wherein after step B3) B5) digesting the second circular nucleic acid fragment with the third restriction enzyme to generate a third set of nucleic acid fragments; B6) modifying the terminal produced by the third restriction enzyme to enable ligation; B7) connecting the ends of said third nucleic acid fragments to produce a third set of circular nucleic acid fragments. 54. The method of claim 53, further comprising repeating steps B1) to B4) for each of a plurality of different second restriction enzymes. 56. The method of any one of claims 53 to 55, wherein each recognition site is located close to the terminus of the first engineered nucleic acid. 59. The method of any one of claims 53-56, wherein each of the recognition sites is located less than 25 nucleotides away from the end of the first engineered nucleic acid. 58. The method of any one of claims 53-57, wherein the recognition site is located 0 to 5 nucleotides away from the end of the first engineered nucleic acid. 59. The method of any of claims 53-58, wherein the second restriction enzyme is a type IIS endonuclease. 60. The method of claim 59, further comprising the step of immobilizing and amplifying the nucleic acid fragment in the second annular nucleic acid fragment on a solid surface prior to step B4). 61. The method of claim 60, wherein said fixing and amplifying step is performed by producing a colony of said nucleic acid fragment in said second annular nucleic acid fragment on said solid surface, said colony being one of said nucleic acid fragments in said second annular nucleic acid fragment. And a plurality of immobilized single-stranded DNA molecules. 62. The method of claim 61, Iii) linearizing the second circular nucleic acid fragment to produce a linear fragment; Ii) providing a solid surface comprising a plurality of such colony primers, each colony comprising a sequence hybridizable with a sequence at the 3 'end of the linear fragment and immobilized at the 5' end on the solid surface; Iii) denaturing the linear fragments to produce single stranded fragments; Iii) annealing the single stranded fragment to the immobilized colony primer; Iii) generating an immobilized double stranded nucleic acid fragment by performing a primer extension reaction using the annealed single stranded fragment as a template; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments with fixed colony primers; Iii) repeating said steps iii) to iii) such that said colonies are formed at each particular location of said solid surface. 62. The method of claim 61, Iii) linearizing the second circular nucleic acid fragment to produce a linear fragment; Ii) mixing the linear fragment with colony primers each comprising a sequence hybridizable with a sequence at the 3 'end of the linear fragment; Iii) implanting the linear fragments and colony primers at 5 'ends on a solid surface to produce fixed linear fragments and fixed colony primers; Iii) denaturing the fixed linear fragment to produce a fixed single stranded fragment; Iii) annealing the fixed single stranded fragment to the fixed colony primer to obtain an annealed single stranded fragment; Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments to the fixed colony primers; And Iii) repeating said steps v) to iii) such that said colonies are produced at each particular site on said solid surface. 62. The method of claim 61, Iii) linearizing the second circular nucleic acid fragment to produce a linear fragment; Ii) the linear fragments are mixed with colony primers each comprising a sequence hybridizable with a sequence at the 3 'end of the linear fragment, wherein the concentration of the colony primers is adjusted to allow amplification of the implanted linear fragments to occur. Doing; Iii) implanting the linear nucleic acid fragments and colony primers at 5 'ends on a solid surface to produce fixed linear fragments and immobilized colony primers; Iii) applying the amplification solution containing polymerase and nucleotides to said solid surface such that said colonies are isothermally produced at a specific location on said solid surface, wherein said colonies are produced. . 65. The method of any of claims 62 to 64, wherein the sequencing is Iii) hybridizing a sequencing primer to the colony; Ii) performing primer extension with one labeled nucleotide; Iii) detecting the amount of labeled nucleotides incorporated into each of the extended primers at said location; And Iii) repeating steps ii) and iii) to determine a portion of the sequence of each colony. 66. The method of claim 65, wherein the labeled nucleotides are nucleotides labeled with fluorescence, and wherein the detection involves detecting fluorescence intensity of the labeled nucleotides. The method of claim 1, wherein determining the restriction sequence tag set B1) linking said restriction fragment in said set of restriction fragments with a first engineered nucleic acid comprising a predetermined nucleotide sequence comprising a recognition site of a second restriction enzyme with a first restriction enzyme to link said first nucleic acid fragment set Obtaining; B2) digesting the first nucleic acid fragment with a second restriction enzyme to obtain a second set of nucleic acid fragments; B3) joining the ends of the second restriction fragment to generate a first set of circular nucleic acid fragments; B4) sequencing at least a portion of each of said fourth nucleic acid fragments to determine said set of restriction sequence tags. The method of claim 53, wherein after step B3) B5) digesting the first cyclic nucleic acid fragment with the third restriction enzyme which is different from the first restriction enzyme and the second restriction enzyme to generate a third set of nucleic acid fragments; B6) modifying the terminal produced by the third restriction enzyme to enable ligation; And B7) connecting the ends of said third nucleic acid fragments to produce a second set of circular nucleic acid fragments. 68. The method of claim 67, further comprising repeating steps B1) to B4) for each of a plurality of different second restriction enzymes. 70. The method of claim 69, further comprising the step of immobilizing and amplifying the nucleic acid fragment in the first annular nucleic acid fragment on a solid surface prior to step B4). 71. The method of claim 70, wherein the fixing and amplifying step is performed by producing colonies of the nucleic acid fragments in the first annular nucleic acid fragment on the solid surface, each of the colonies being in the nucleic acid fragments in the first annular nucleic acid fragment. A method comprising a plurality of immobilized single stranded DNA molecules. The method of claim 71 wherein Iii) linearizing the first circular nucleic acid fragment to produce a linear fragment; Ii) providing a solid surface comprising a plurality of such colony primers, each colony comprising a sequence hybridizable with a sequence at the 3 'end of the linear fragment and immobilized at the 5' end on the solid surface; Iii) denaturing the linear fragment to produce a single stranded fragment; Iii) annealing the single stranded fragment to the immobilized colony primer; Iii) generating an immobilized double stranded nucleic acid fragment by performing a primer extension reaction using the annealed single stranded fragment as a template; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments with fixed colony primers; Iii) repeating said steps iii) to iii) such that said colonies are formed at each particular location of said solid surface. The method of claim 71 wherein Iii) linearizing the first circular nucleic acid fragment to produce a linear fragment; Ii) mixing the linear fragment with colony primers each comprising a sequence hybridizable with a sequence at the 3 'end of the linear fragment; Iii) implanting the linear fragments and colony primers at 5 'ends on a solid surface to produce fixed linear fragments and fixed colony primers; Iii) denaturing the fixed linear fragment to produce a fixed single stranded fragment; Iii) annealing the fixed single stranded fragment to the fixed colony primer to obtain an annealed single stranded fragment; Iii) performing a primer extension reaction using the annealed single stranded fragment as a template to generate an immobilized double stranded nucleic acid fragment; Iii) denaturing the immobilized double stranded nucleic acid fragments to produce immobilized single stranded fragments; Iii) annealing the fixed single stranded fragments to the fixed colony primers; And Iii) repeating said steps v) to iii) such that said colonies are produced at each particular site on said solid surface. The method of claim 71 wherein Iii) linearizing the first circular nucleic acid fragment to produce a linear fragment; Ii) the linear fragments are mixed with colony primers each comprising a sequence hybridizable with a sequence at the 3 'end of the linear fragment, wherein the concentration of the colony primers is adjusted to allow amplification of the implanted linear fragments to occur. Doing; Iii) implanting the linear fragments and colony primers at 5 'ends on a solid surface to produce fixed linear fragments and fixed colony primers; Iii) applying the amplification solution containing polymerase and nucleotides to said solid surface such that said colonies are isothermally produced at a specific location on said solid surface, wherein said colonies are produced. . 75. The method of any of claims 72-74, wherein the sequencing is Iii) hybridizing a sequencing primer to the colony; Ii) performing primer extension with one labeled nucleotide; Iii) detecting the amount of labeled nucleotides incorporated into each of the extended primers at said location; And Iii) repeating steps ii) and iii) to determine a portion of the sequence of each colony. 76. The method of claim 75, wherein said labeled nucleotides are nucleotides labeled with fluorescence, and said detection involves detecting the fluorescence intensity of said labeled nucleotides. 77. The method of any one of claims 1 to 76, further comprising the step of digesting said restriction fragment set with a plurality of different first restriction enzymes in step A). 78. The method of any one of claims 1-77, wherein each said group consists of restriction sequence tags wherein at least 60% are homologous. 79. The method of claim 78, wherein each said group consists of restriction sequence tags wherein at least 70% are homologous. 80. The method of claim 79, wherein each said group consists of restriction sequence tags that are at least 80% homologous. 81. The method of claim 80, wherein each said group consists of restriction sequence tags that are at least 90% homologous. 84. The method of claim 81, wherein each said group consists of restriction sequence tags that are at least 99% homologous. A) by the method of any of claims 1-82, determining a set of restriction sequence tags for each population of organisms having one or more organisms for each of a plurality of different phenotypes; B) A method of determining genome-wide sequence variation among a plurality of different phenotypes, comprising comparing said set of restriction sequence tags in living organisms of different phenotypes to determine one or more sequence variations associated with different phenotypes. . 84. The method of claim 83, further comprising mapping the at least one restriction sequence tag to the genomic sequence of the organism after step B) to identify the genomic location of the at least one restriction sequence tag. . 77. The method of any one of claims 45-52, 55-66, and 69-76, wherein the plurality of different second restriction enzymes comprise at least three different restriction enzymes. Characterized in that. A) by the method of claim 85, determining a set of restriction sequence tags for each population of organisms having one or more organisms for each of a plurality of different phenotypes; B) A method of determining genome-wide sequence variation among a plurality of different phenotypes, comprising comparing said set of restriction sequence tags in living organisms of different phenotypes to determine one or more sequence variations associated with different phenotypes. . 87. The method of claim 86, further comprising mapping the at least one restriction sequence tag to the genomic sequence of the organism after step B) to identify the genomic location of the at least one restriction sequence tag. . 77. The method of any one of claims 45-52, 55-66, and 69-76, wherein the plurality of different second restriction enzymes comprises at least 10 different restriction enzymes. Characterized in that. A) by the method of claim 88, determining a set of restriction sequence tags for each population of organisms having one or more organisms for each of a plurality of different phenotypes; B) A method of determining genome-wide sequence variation among a plurality of different phenotypes, comprising comparing the set of restriction sequences tags in living organisms of different phenotypes to determine one or more sequence variations associated with different phenotypes. . 90. The method of claim 89, further comprising mapping the at least one restriction sequence tag to the genomic sequence of the organism after step B) to identify the genomic location of the at least one restriction sequence tag. . 83. The method of any one of the preceding claims, wherein said one or more individual living beings are humans. 84. The method of any one of the preceding claims, wherein each set of restriction fragments comprises at least ten different restriction fragments. 84. The method of any one of the preceding claims, wherein each set of restriction fragments comprises at least 100 different restriction fragments. 84. The method of any one of the preceding claims, wherein each set of restriction fragments comprises at least 1000 different restriction fragments. 84. The method of any one of the preceding claims, wherein each set of restriction fragments comprises at least 10,000 different restriction fragments. 83. The method of any one of the preceding claims, wherein each set of restriction fragments comprises at least 100,000 different restriction fragments. 83. The method of any one of the preceding claims, wherein each set of restriction fragments comprises at least 10 ⁶ different restriction fragments. 84. The method of any one of the preceding claims, wherein each of the set of restriction fragments comprises at least 10 ⁷ different restriction fragments. 84. The method of any one of the preceding claims, wherein each set of restriction fragments comprises at least 10 ⁸ different restriction fragments. 100. The method of any one of claims 1 to 99, wherein step I) is performed on one individual. 101. The method of any one of claims 1-100, wherein said step II) of classifying a restriction sequence tag further comprises comparing said restriction sequence tag with a reference sequence. 102. The method of claim 101, wherein said reference sequence comprises a genomic sequence of an organism.