JP2007515956A - Improved selective ligation and amplification assay - Google Patents

Abstract

An improved assay for identifying and distinguishing one or more single nucleotide polymorphisms in one or more target sequences of nucleic acids comprises three or more primers in a single tube reaction system, two of which are 1 Bind to the target nucleic acid sequence adjacent to the SNP such that the 3 ′ end of the first primer is adjacent to the 5 ′ end of the second primer, the two primers are selectively ligated, Amplified by a third primer to generate exponentially complementary strands of one or more target sequences. The other strands of the one or more target sequences are exponentially amplified by one or more hybridizable probes, each labeled with a different fluorophore, and the fluorophore-labeled hybridizable probe becomes a target nucleic acid product. Is quenched until the uptake and its amplification.

Description

  The present invention relates to assays for amplifying and identifying target sequences of nucleic acids associated with combined ligation and amplification protocols, and the use of nanoliter sampling arrays to perform such assays.

  Genetic variation is increasingly associated with a number of disease states and predisposition to illness, including cancer, multiple sclerosis, autoimmune disease, cystic fibrosis and schizophrenia. The ability to quickly and inexpensively identify genetic variation greatly facilitates diagnosis, risk assessment, and prognostic decisions about such diseases and predisposition to these diseases.

  One possibility for identifying genetic variation combines the selective ligation and amplification techniques disclosed in US Pat. Nos. 6,099,028 to Bhatnagar et al. And US Pat. Including that. Both patents disclose the use of at least three primers, two of which are complementary to the adjacent region at the 3 ′ end of one strand of the target nucleic acid sequence that can be ligated after hybridization and then extended. is there. In Todd et al., The third primer is complementary to the random sequence at the 3 ′ end of the downstream primer (linked to the upstream primer) and is identical to the random sequence at the 5 ′ end of the first primer. Is a random sequence. In Bhatnagar et al., The third primer is complementary to the upstream primer and is also complementary to the opposite strand of the target sequence. In both cases, there must be complementarity at the 3 'end of the third primer to cause amplification.

A target nucleic acid sequence is amplified using a thermo-stable polymerase, and both ligation and amplification reactions can be performed in the same reaction mixture. There can be any gap between adjacent primers, which can be filled by the polymerase and allow for successful ligation of adjacent primers. Such a system allows the identification of gene variability in the target nucleic acid sequence and the identification of multiple alleles.
US Pat. No. 5,593,840 US Pat. No. 6,245,505

(Summary of the Invention)
In a first embodiment of the invention, an improved assay of the type for amplifying a specific target nucleic acid sequence is provided, wherein the target sequence comprises an internal SNP of interest, the assay comprising a target sequence, Ligable first and second primers each having at least a portion substantially complementary to the first and second segments of the target sequence, and substantially to the random sequence segments of the first and second primers A selective ligation and amplification method of the type using a controlled temperature reaction mixture comprising a third primer that is complementary in nature, wherein the improvement is: to hybridize to a target nucleic acid sequence having a SNP of interest Differentiate between one or more SNPs in one or more target sequences of nucleic acids in a single tube reaction system using two unique probes designed It said method comprising, each hybridizable probe having a different fluorescent tag is quenched to the probe incorporation into the amplified target nucleic acid product.

  In some embodiments of an improved assay of the type for amplifying a specific target nucleic acid sequence comprising one or more SNPs of interest that are not at the end of the target sequence, the assay comprises a target A first primer having at least a portion of its 3 ′ end that is substantially complementary to the first segment at the first end of the target sequence, a second segment at the second end of the target sequence A second primer having at least a portion of its 5 'end that is substantially complementary to the 5' end of the second primer adjacent to the 2-4 bases of the 3 'end of the first primer Or in which the nucleotide complementary to the SNP of the target sequence is present either at the 3 ′ end of the first primer or at the 5 ′ end of the second primer And a third primer that is substantially complementary to a random sequence segment at the 3 ′ end of the first primer and to a substantially similar sequence at the 5 ′ end of the first primer, at least A type of selective ligation and amplification method using a thermocycling reaction mixture comprising four different nucleotide bases, a thermostable polymerase and a thermostable ligase, wherein the improvement is to hybridize to a target nucleic acid sequence having the SNP of interest. Discriminating between one or more SNPs in one or more target sequences of a nucleic acid in a single tube reaction system using two unique probes designed to soy, each hybridizable probe comprising: Has different fluorescent tags that are quenched until incorporation of the probe into the amplified target nucleic acid productA first hybridizable probe having a first fluorescent tag is obtained from a linked first primer-second primer product having a first SNP of interest at the 3 ′ end of the first primer. Having a unique random sequence that hybridizes to the first amplified target nucleic acid generated by the three primers, whereby the first hybridizable probe is incorporated into the amplified opposite strand target nucleic acid product. Thus, a first fluorescent signal is given. A second hybridizable probe with a second fluorescent tag is derived from a different linked first primer-second primer product having a second SNP of interest at the 3 ′ end of the first primer. A unique random sequence that hybridizes to the second amplified product produced by the third primer, and the second hybridizable probe is thereby incorporated into the amplified opposite strand target nucleic acid product. Thus, a second fluorescent signal is given.

  In a preferred embodiment, the random sequence of the first and second hybridizable probes is a unique sequence, and thus the specific incorporation of each hybridizable probe into the amplified target nucleic acid is hybridized. Occurs preferentially after ligation of the first primer-second primer product with the particular SNP of interest designed to detect possible probes. Upon incorporation of the hybridizable probe into the amplified product, fluorescence occurs, allowing detection of the amplified product to be distinguished from non-specific background products. In addition, the random sequence of the third primer may also be present in the presence of human or other species chromosomes to a sufficiently low level that such non-specific products will not interfere with the detection of amplified products with the SNP of interest. Is a unique sequence optimized for PCR to reduce non-specific amplification products that may occur in

  Alternatively, the two hybridizable probes do not contain a fluorescent tag, but are merely additional primers designed to distinguish different ligation products with different SNPs of interest. The amplified product with the SNP of interest is then detected using an additional hybridizable probe, similar to the additional primer, but developed so as not to interfere with the amplification. These hybridizable probes have fluorescent tags or, alternatively, each have a different fluorescent tag, which fluoresces when hybridized to the amplified product, thereby allowing amplification of the amplified product. Enable detection.

  In another embodiment of the invention, an improved assay of the type for amplifying a specific target nucleic acid sequence is provided, wherein the target sequence comprises a SNP of interest that is not at the end of the target sequence; The assay includes a target sequence, a first primer having at least a portion of its 3 ′ end that is substantially complementary to a first segment at the first end of the target sequence, at the second end of the target sequence. A second primer having at least a portion of its 5 'end that is substantially complementary to a second segment, wherein the 5' end of the second primer is adjacent to the 3 'end of the first primer. Wherein the nucleotide complementary to the SNP of the target sequence is present at either the 3 ′ end of the first primer or the 5 ′ end of the second primer, and of the second primer A third primer that is substantially complementary to a random sequence segment at the 'end and to a substantially similar sequence at the 5' end of the first primer, at least four different nucleotide bases, heat A selective ligation and amplification method of the type using a thermocycling reaction mixture comprising a stable polymerase and a thermostable ligase, wherein the improvement is a double-stranded (ds) that fluoresces upon binding to a target sequence Including uniformly detecting the amplified target sequence using a dye specific for binding to DNA. In a preferred embodiment, the random sequence of the third primer is a unique sequence optimized for PCR so that non-specific products do not occur in the presence of chromosomes of humans or other species. In some embodiments, the primer may be deposited in or under a biocompatible material, such as a wax-like coating on the surface of the through hole, by drying the primer after application to the through hole. Here, the biocompatible material can include, for example, a polyethylene glycol (PEG) material.

Alternatively, the assay according to the invention may be performed by a thermostable polymerase lacking 5′-3 ′ exonuclease activity, or a thermostable polymerase lacking 3′-5 ′ exonuclease activity, or 5′-3 ′ and 3 ′. A thermostable polymerase lacking both -5 'exonucleases can be used. Examples of thermostable polymerases lacking 5′-3 ′ exonuclease activity include Stoffel fragments, Isis ^™ DNA polymerase, Pyra ^™ exo (−), DNA polymerase and Q-BioTaq ^™ DNA polymerase. Examples of thermostable polymerases that lack 3′-5 ′ exonuclease activity include Taq polymerase, SurePrime ^™ polymerase, and Q-BioTaq ^™ DNA polymerase. An example of a thermostable polymerase that lacks both 5′-3 ′ and 3′-5 ′ exonuclease activity is Q-BioTaq ^™ DNA polymerase. Suitable dyes are SYBR® Green I and SYBR® Green II, YOYO®-1, TOTO®-1, POPO®-3, ethidium bromide, or fluorescent Any other dye that allows for rapid sensitivity detection of target nucleic acid sequences amplified using

  In another embodiment, a nanoliter sampling array is provided that includes a first platen having at least one hydrophobic surface and having a dense microfluidic array of hydrophilic through-holes. In this particular embodiment, each through hole has at least a first portion having at least a portion of its 3 ′ end that is substantially complementary to the first segment at the first end of the potential nucleic acid target sequence. And a second primer having at least a portion of its 5 ′ end that is substantially complementary to the second segment at the second end of the potential nucleic acid target sequence, The 5 ′ end is adjacent to the 3 ′ end of the first primer upon binding to a potential nucleic acid target sequence.

  In addition, the sampling array can further include a second platen having at least one hydrophobic surface and having a dense microfluidic array of hydrophilic through holes, wherein the first and The second platen is fixedly coupled so that the respective through holes are aligned.

  In yet another embodiment, a method for identifying a SNP in a target sequence of a nucleic acid is provided, the method providing a first sample platen having a high-density microfluidic array of through holes, each through hole being a target sequence. A first primer having at least a portion of its 3 ′ end that is substantially complementary to the first segment at the first end of the first sequence, substantially the second segment at the second end of the target sequence A second primer having at least a portion of its 5 ′ end that is complementary, the 5 ′ end of the second primer is adjacent to the 3 ′ end of the first primer, and the second primer Having a third primer that is substantially complementary to a random sequence segment at the 3 ′ end and to the 5 ′ end of the first primer; A sample containing a target sequence of nucleic acid having a SNP is introduced into the array, a reagent is introduced into a through-hole in the array, the reagent comprising a thermostable polymerase, a thermostable ligase, and at least four different nucleotide bases And subjecting the array to thermal cycling and then detecting the amplified target sequence. In a preferred embodiment, primers 1 and 2 are in the target strand SNP located at either the 3 ′ end of the 5 ′ primer (first primer) or the 5 ′ end of the 3 ′ primer (second primer). Designed to have possible matches against. If the first and second primers are adjacent to each other and hybridize to a target strand close to the SNP, primer ligation occurs only if there is a successful match for the SNP by one of the primers. . In this way, ligation is selective and so selective amplification of the desired target sequence including the SNP of interest also occurs. As described above, in some embodiments, the primer is in or on a biocompatible material, such as a wax-like coating on the surface of the through hole, by drying the primer after application to the through hole. The biocompatible material can include, for example, a polyethylene glycol (PEG) material.

In addition, the method of identifying a SNP in a target sequence of a nucleic acid additionally uses a thermostable polymerase that lacks 5′-3 ′ exonuclease activity and then fluoresces upon binding to the target nucleic acid. Detecting the amplified target sequence with a dye specific for binding to the strand (ds) DNA can be included. Alternatively, detection results in amplified target sequences having different melting curves, and a first primer designed to distinguish individual amplified target sequences by differences in melting temperature (T _m ) And a probe specific for hybridization across the ligation junction formed between the first primer and the second primer after binding to the target sequence. Wherein the probe specific for hybridizing across the ligation junction has a fluorescent group and a fluorescent modifying group, or alternatively for hybridizing to a fluorescent group and a region of the target sequence. Using a probe containing a specific fluorescent modifying group, where upon extension of the probe, the fluorescent modifying group is cleaved and the fluorescent group is Emit light. In addition, detection can be accomplished using a probe specific for hybridizing to any unique sequence in the amplified target nucleic acid sequence, which fluoresces and is amplified upon hybridization. It has a fluorescent group and a fluorescent modifying group so that the target nucleic acid can be detected.

Another means of detection involves the use of an amplification primer that matches the random sequence of primer 2, where the primer is fluorescent only when incorporated into the PCR product, as is the Lux ^™ primer known in the art. Labeled with a fluorescent group that emits. In such embodiments, the fluorescent group is a double-stranded product such that prior to incorporation, the sequence in the primer / probe binds to the complementary sequence in the primer / probe containing the fluorescent group and quenches the fluorescent group. Quenched by the second structure before incorporation into. In another embodiment, primers 1 and 2 are generated only after primers 1 and 2 are ligated and amplified when hybridized to the amplified target sequence, such that they fluoresce and have fluorescence resonance energy. A mobile (FRET) partner.

  Yet another embodiment provides a kit for use in the identification of amplified target nucleic acid sequences, the kit having a hydrophobic surface and a dense microfluidic array of hydrophilic through-holes Includes platen. In the array of kits, each through-hole has at least a first portion having at least a portion of its 3 ′ end that is substantially complementary to the first segment at the first end of the potential nucleic acid target sequence. And a second primer having at least a portion of its 5 ′ end that is substantially complementary to a second segment at the second end of the potential nucleic acid target sequence, the 5 ′ of the second primer The end is adjacent to the 3 ′ end of the first primer upon binding to a potential nucleic acid target sequence. The kit also includes a reagent platen having a high-density microfluidic array of through holes, each through hole against a random sequence segment at the 3 ′ end of the second primer and at the 5 ′ end of the first primer. Contains a third primer that is substantially complementary to a substantially similar sequence, at least four different nucleotide bases, a thermostable polymerase and a thermostable ligase. In the kit of this embodiment, the reagent platen has a structural geometry that corresponds to the sample platen, thereby allowing delivery of the reagent components and the target nucleic acid sample to the primers in the sample platen. In other embodiments, the thermostable polymerase can lack 5'-3 'exonuclease activity.

  The foregoing features of the invention will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:

(Detailed description of specific embodiment)
Definition. As used herein and in the appended claims, the following terms should have the meanings indicated, unless otherwise required.

  “Target nucleic acid”, “target nucleic acid sequence” or “latent target nucleic acid sequence” means any prokaryotic or eukaryotic DNA or RNA, including from plants, animals, insects, microorganisms and the like. It can be isolated or present in a sample containing the nucleic acid sequence in addition to the target nucleic acid sequence to be amplified. The target nucleic acid sequence can be located within a longer nucleic acid sequence than that of the target sequence. The target nucleic acid sequence can be obtained synthetically or enzymatically or can be isolated from any organism by methods well known in the art. Particularly useful sources of nucleic acids are derived from biological tissues or blood samples, nucleic acids present in self-replicating vectors, and nucleic acids derived from viruses and pathogenic organisms such as bacteria and fungi. Also particularly useful are related to disease states, such as those caused by chromosomal rearrangements, insertions, deletions, translocations and other mutations, those caused by oncogenes, and those associated with cancer Target nucleic acid sequence.

  “Selected” means that a target nucleic acid sequence having the desired characteristics is located and the probe is built around an appropriate segment of the target nucleic acid.

  “Probes” or “primers” have the same meaning herein, ie, nucleic acid oligonucleotide sequences that are single stranded. The term oligonucleotide includes DNA, TNA and PNA.

  A probe or primer is “substantially complementary” to a target nucleic acid sequence if it hybridizes to the sequence under regenerative conditions that allow target-dependent ligation or extension. Regeneration depends on specific base pairing between AX (where X is T or U) and GC bases to form a double-stranded duplex structure. Thus, the primer sequence need not reflect the exact sequence of the target nucleic acid sequence. However, if an exact copy of the target sequence is desired, the primer should reflect the exact sequence. Typically, a “substantially complementary” primer will contain at least 70% or more bases complementary to the target nucleic acid sequence. More preferably, 80% or more of the bases are complementary, and even more preferably, more than 90% of the bases are complementary. In general, the primer should hybridize to the target nucleic acid sequence at the end to be ligated or extended to allow target-dependent ligation or extension.

  Primers can be RNA or DNA, are analogs of normally incorporated bases, or some modified nitrogen modified by attaching a label, or a linker arm suitable for attaching the label A basic base can be contained. Inosine can be used where the target sequence is unknown or where it can be denatured. The oligonucleotide should be long enough to allow hybridization of the primer to the target sequence and to allow amplification to proceed. They are preferably 15 to 50 nucleotides long, more preferably 20 to 40 nucleotides long, even more preferably 25 to 35 nucleotides long. The nucleotide sequence of the primer will vary depending on the target sequence to be amplified, both in content and length.

  It is contemplated that a primer can include one or more oligonucleotides that contain a sequence that is substantially complementary to a target nucleic acid sequence. Thus, under lower stringency conditions, each of the oligonucleotide primers will hybridize to the same segment of the target sequence. However, under increasing stringent conditions, only the oligonucleotide primer that is most complementary to the target nucleic acid sequence will hybridize. It is known to those skilled in the art that the stringency of hybridization conditions generally depends on temperature, solvent, ionic strength and other parameters. One of the most easily controlled parameters is temperature, and the conditions for selective ligation and amplification are similar to those of the PCR reaction, so that one skilled in the art can achieve the desired level of stringency. The appropriate conditions required can be determined.

  Primers suitable for use in the present invention may be from any method known in the art, including chemical or enzymatic synthesis, or by cleavage of larger nucleic acids using non-specific nucleic acid cleavage chemicals or enzymes. Alternatively, it can be induced by using site-specific restriction endonucleases.

  In order for the ligase of the present invention to ligate primers together, the primers used are preferably phosphorylated at their 5 'ends. This can be achieved by any known method in the art, including the use of T4 polynucleotide kinase. Primers can be phosphorylated in the presence of unlabeled or radiolabeled ATP.

  The term “four different nucleotide bases” refers to deoxythymidine triphosphate (dTTP), deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate when the context is DNS. Means (dGTP), and when the context is RNA, means uridine triphosphate (UTP), adenosine triphosphate (ATP), cytidine triphosphate (CTP), and guanosine triphosphate (GTP) . Alternatively, dUTP, dITP (deoxyinosine triphosphate), rITP (riboinosine triphosphate) or any other modified base can replace any of the four nucleotide bases, or the reaction mixture It is contained together with the four nucleotide bases and can be incorporated into the amplified strand. The amplification step is performed in the presence of at least four deoxynucleotide triphosphates (dATP, dCTP, dGTP and dTTP), or modified nucleoside triphosphates to generate a DNA strand, or four ribonucleoside triphosphates. Performed in the presence of acids (ATP, CTP, DTP and UTP0) or modified ribonucleoside triphosphates to generate RNA strands from primer extension.

  The term “appropriate detection of the desired amplified product” means the detection of at least a 2-fold increase in the desired amplified target strand over the competing linear product.

  The term “target sequence detectable beyond the linearly amplified product” means that the target sequence is amplified at least twice that of the competing linearly amplified unligated primer product. .

  As used herein, the term “random sequence” is selected so that it does not bind to the target sequence or to other sequences that would be expected to be present in the target sequence or test sample. Means a sequence.

  As used herein, the term “biocompatible material” means that the material does not prevent a biological process, such as an enzymatic reaction, from occurring when the biocompatible material is present. And does not exclude the biological activity or required secondary, tertiary or quaternary structure of biomolecules such as proteins and generally means not incompatible with biological processes and molecules.

  As used herein, the term “first and second primers that can be linked upon binding to a nucleic acid target sequence” means that the first and second primers optionally add polymerase activity to fill the gap. Subjecting the hybridized first and second primers to appropriate enzymatic or non-enzymatic ligation conditions results in a single ligation of the first and second primers enzymatically or non-enzymatically. A potential target nucleic acid having a 3 ′ end of the first primer adjacent to or within an approximately 1- to 4-nucleotide gap at the 5 ′ end of the second primer, such that Means to bind to.

  As used herein, the term “polymerase” means any oligomeric synthase, including polymerases, helicases, and other protein fragments capable of polymerizing the synthesis of oligomers.

  As used herein, the term “controlled temperature reaction mixture” allows temperature control of the reaction, including thermocycle devices, isothermal devices, or temperature-controlled environments such as water, oil and sand baths, incubation chambers, etc. By any other means known to do is meant any reaction mixture whose temperature is controlled.

  Through detection of the amplified target sequence using a dye specific for binding to double-stranded DNA that fluoresces upon binding to the target sequence according to the present invention, a single nucleotide polymorphism that is not at the end of the target sequence ( A general assay for identifying (SNP) is described later and illustrated in FIGS. The assay is performed in a single reaction chamber or container, in a series of reaction chambers or containers, in a nanoliter sampling array with a high-density microfluidic array of hydrophilic through-holes, or in a kit containing such an array and the necessary reagents. It can be carried out. Detection can be uniform and a polymerase lacking 5'-3 'exonuclease activity, or a polymerase lacking 3'-5' exonuclease activity or a polymerase lacking both exonuclease activities can be used.

The assay can be performed with three (P1, P2, P3) or more (AB, CD, FE, D ′) primers to simultaneously detect one or more SNPs in a single target Is possible. In some versions of the assay, a nucleotide complementary to the target nucleotide SNP is present at or near the 5 ′ end of the second primer P2. In other versions, a nucleotide complementary to the target nucleotide SNP is present at or near the 3 ′ end of the first primer P1. In other versions, there are more than one first and second primers, and these first and second primers were amplified individually due to their individual and distinct T _m assays. Designed to produce amplified target sequences with different melting temperatures so that the target sequences can be identified.

  The assay can be performed with first and second primers that include degenerate base-pairing positions that allow for hybridization of variable regions in the target sequence adjacent to the SNP, thus allowing for general flexibility of the assay. To expand sex and usability.

  Primers 1 and 2, corresponding to the 5 'and 3' linked primers, respectively, can be sufficiently complementary or partially complementary to the target sequence. Primer 3 is a generic primer complementary to a random sequence (RS) located at the end of primer 1 and / or 2 (see FIGS. 1 and 2). The 3 ′ end of primer 1 and the 5 ′ end of primer 2 can hybridize immediately adjacent to each other on the target sequence, or can be separated, or separated by a gap or one or more nucleotides therebetween, It can hybridize to the sequence (see FIGS. 1-2 and 4-5). Primer 1 or 2 includes a variant base at or near the 3 ′ end (P1) or 5 ′ end (P2) to allow the primer to bind to SNPS in the target sequence (FIGS. 1-2). reference). There is also a 3'-hydroxyl group on P2 to facilitate polymerase extension prior to enzymatic or non-enzymatic linkage between P1 and P2, or linkage (to fill either gap). In addition, the 5 'end of P2 can be modified to prevent unwanted ligation to fragments other than P1.

  Similarly, the 5 'end of P1 can be phosphorylated to facilitate ligation with P2, and the 3' end of P1 can be modified to prevent ligation to fragments other than P2. Amplification of the target nucleic acid is illustrated in FIGS. If necessary, the target nucleic acid and primer are denatured to allow selective extension of the ligation of primers P1 and P2, and temperature is used to anneal it.

  Detection of single-stranded ligation products is done using several strategies, some have occurred using hybridization probes that hybridize to single-stranded amplification products, or both sense and non-sense strands of ligation products A dye specific for binding to the double stranded DNA produced after the extension and amplification of the DNA is used. Other detection strategies use molecular beacons attached to hybridizable probes. And yet other detection strategies use the use of FRET pairs with hybridizable probes. In some assays, a fluorescent dye is simply added to the reaction mixture and the change in fluorescence intensity is monitored to detect the ligated product. In other assays, a hybridizable probe is added after generation of a ligation product that is specific for the ligation product and also contains a molecular beacon or a fluorescent group and a fluorescent modifying group. The hybridizable probe can be attached to the extended ligation product, the rest being quenched by the fluorescent modifying group until extended to the amplified product, where the fluorescent group fluoresces and amplified The target sequence can be detected (see FIG. 4), or the hybridizable probe can be specific for hybridizing across the ligation junction, where the probe is quenched again until after hybridization (see FIG. 5). ). In the assay described in FIG. 4, one or more hybridizable probes can be used, each distinct fluorophore, and a unique that hybridizes to and amplifies each of the one or more target nucleic acid sequences. Which allows a large number of SNPs to be detected in a single tube reaction system.

  Also, any of the assays can be performed on a nanoliter sampling array. The nanoliter array can include one or more platens having a dense microfluidic array of at least one hydrophobic surface and hydrophilic through holes. The inner surface of the through hole can be coated with a biocompatible material, such as a wax-like polyethylene glycol material, or other biocompatible material. The primer is applied to the through hole and then dried before or after application of the biocompatible material coating, thereby depositing the primer on, within, or below the biocompatible material. The target nucleic acids and reagents for the process used in the selective ligation and amplification assay are liquid in the sample through hole using capillary action, with a typical volume of the sample through hole ranging from 0.1 picoliter to 1 microliter. Can be loaded in form. The inner surface of the through hole can also have a hydrophilic surface, or it can be coated with a porous hydrophilic material, or as described above, coated with a biocompatible material such as PEG, The suction power of the through hole can be increased, the liquid sample can be attracted, and the load can be assisted.

  A kit for performing the assay can also be prepared, which includes one or more of the sample platens described above, the primer is attached within the hydrophilic sample through hole of the microfluidic array, and is also required for selective ligation and amplification assays Various reagents may also be included. The target nucleic acid sequence can then be added if desired to perform the assay. If the kit is not already provided, the enzymes necessary to perform the ligation and amplification reaction can be added along with the target nucleic acid sequence.

Example 1 Uniform Detection of Amplified Target Sequence
Homogeneous detection of the amplified target sequence can be performed using a dye specific for binding to double stranded DNA or RNA. Primers P1 and P2, respectively, upstream and downstream primers do not participate in the amplification of the target sequence, but rather are responsible for identifying the target sequence containing the SNP. If either primer P1 or P2 contains a match to the SNP of interest in the target sequence, ligation of P1 and P2 occurs, then primer P3, a general extension primer, amplifies the P1-P2 product. As a result, the concentrations of primers 1 and 2 are preferably optimized and adjusted so as not to interfere with the exponential amplification of the target sequence so that only linear amplification of competing non-target sequences occurs. Examples of ds-DNA and / or RNA specific dyes that can be used are SYBR® Green I and SYBR® Green II, YOYO®-1, TOTO®-1, POPO (Registered trademark) -3 (see Appendix A attached hereto), ethidium bromide (EtBr), and any other dye that provides adequate sensitivity and ease of detection of the desired amplified product .

In a special embodiment, the sample target sequence of the nucleic acid optionally containing a single nucleotide polymorphism is optionally a 3 ′ end substantially complementary to the first segment at the first end of the target sequence, ie the first end of the target sequence A first upstream primer having at least a portion; a second downstream primer having at least a portion at its 5 ′ end that is substantially complementary to a second segment at the second end of the target sequence; The 5 ′ end of the first primer is adjacent to the 3 ′ end of the first primer, where the nucleotide complementary to the SNP of the target sequence is the 3 ′ end of the first primer or the second primer. To a random sequence segment present at any of the 5 ′ ends and at the 3 ′ end of the second primer, and to the 5 ′ end of the first primer Mixing a third common extension primer that is substantially complementary to substantially similar sequences definitive. In addition, at least four different nucleotide bases, a thermostable polymerase and a thermostable ligase are included in the reaction mixture, and the thermostable polymerase is preferably such as a Stoffel fragment (see Appendix B attached hereto). It lacks 5′-3 ′ exonuclease activity. Examples of other thermostable polymerases lacking 5′-3 ′ exonuclease activity include Isis ^™ DNA polymerase, Pyra ^™ exo (−) DNA polymerase, and Q-BioTaq DNA polymerase (see Appendix C attached hereto) ). Alternatively, the assay can use a thermostable polymerase that lacks 3′-5 ′ exonuclease activity, or a thermostable polymerase that lacks both 5′-3 ′ and 3′-5 ′ exonuclease activity. Examples of thermostable polymerases lacking 3′-5 ′ exonuclease activity include Taq polymerase, SurePrime ^™ polymerase, and Q-BioTaq ^™ DNA polymerase (Id.). An example of a thermostable polymerase that lacks both 5′-3 ′ and 3′-5 ′ exonuclease activity is Q-BioTaq DNA polymerase (Id.). Addition of a dye specific for ds-DNA such as SYBR® Green I, or RNA specific for SYBR® Green II, will result in fluorescence emission of the dye-bound nucleic acid product at ˜520 nm. Monitoring allows detection of the amplified product (see Appendix D attached here).

As can be seen in FIG. 1-A, the target nucleic acid can contain a SNP within the target sequence. Upon denaturation, primer 1 (P1) and primer 2 (P2) bind to the 3′-5 ′ strand of the target sequence adjacent to the SNP. There may be several (approximately 2 to 4) base gaps between the 3 ′ end of P1 and the 5 ′ end of P2, or no gap. In FIG. 1-B1, the base complementary to the SNP of the target sequence is at the 3 ′ end of P1. Alternatively, the base complementary to the SNP of the target sequence may be at the 5 ′ end of P2, as shown in FIG. 1-B2. The third primer (P3) binds to and extends the primer product to which P3 is linked after the ligation of P1 and P2, thereby extending the complementary strand (5′-3 ′ strand of the target sequence). A random sequence (RS) complementary to the random sequence at the 3 ′ end of P2. As discussed above, a competitive reaction can occur such that primer P3 binds to primer P2 and extends this sequence to produce a linear product based on the P2 sequence. Preferably, the concentrations of primers P1 and P2 are adjusted to minimize competing linear reactions. As shown in FIG. 1-C, the unlinked primer P1 extends the 3′-5 ′ strand of the target sequence.
In another preferred embodiment shown in FIGS. 2A-E, the first primer (P1) also has a random sequence at the 5 ′ end. When a primer containing a complement to SNP, either P1 at the 3 ′ end or P1 at the 5 ′ end (FIG. 1-B) binds to the target strand (see FIG. 2-B), primers 1 and P2 are ligated. The third primer (P3) then binds to the 3 ′ end of the ligated P1-P2 product, yielding (3′-5 ′) P3-amplified strand (FIG. 2-C). At this point, primer-P3 now also binds to the (3′-5 ′) P3-amplification product, yielding another (5′-3 ′) amplification product (see FIG. 2-D). Both target strands have now been generated and can continue to yield an exponentially amplified target sequence (FIGS. 2-E1 and 2-E2). In addition, detection with fluorescent dyes such as SYBR® Green I (SGI) can be performed at temperatures above the T _m of linear products, ie any non-exponentially generated product. Capable of removing competing signals from any dye bound to the linear product. SYBR® Green I and other dyes that bind to double-stranded nucleic acids do not bind to nucleic acids above their T _m . This is because nucleic acids denature at these elevated temperatures. As seen in the carton of FIG. 3, a dye such as SYBR® Green I binds to a double-stranded amplified target nucleic acid with a concomitant large increase in fluorescence. Although SGI is shown in FIG. 3 to intercalate to the amplified target ds-nucleic acid, FIG. 3 suggests either an actual structure or an actual aspect of binding to the SGI with the ds-nucleic acid. There is nothing intended to do.

Alternatively, the use of a molecular beacon probe with a fluorescent group at one end and a fluorescence quenching group at the other end can be used. In this system, the molecular beacon remains quenched until it binds to the amplified product (see, eg, Appendix E attached hereto). Because molecular probes are typically hairpins with a fluorescent group close to the fluorescence quenching group until the probe binds to the target amplification product (unfolds the hairpin structure and separates the fluorescent group from the quenching group). This is because it is in a three-dimensional configuration. Examples of fluorescence quenching groups suitable for embodiments of the present invention include dark quencher dabcyl, and Eclipse ^™ Quencher from Epoch (Id.). Examples of suitable fluorescent groups that can be used in accordance with the present invention are Epoch's Ykima Yellow ^™ and Redmond Red ^™ (supra), and any other that can quench its fluorescence to an appropriately located quencher molecule. Of a suitable fluorescent dye.

  In another embodiment, real-time amplification can be measured using a TaqMan® probe that is homologous to the internal sequence of the target nucleic acid and has a fluorescent 5 ′ end and a quencher 3 ′ end. . During PCR amplification and extension, the quencher molecule is removed from the probe by 5'-exonuclease activity, causing the quencher molecule at the 3 'end of the probe to release a fluorescent reporter molecule from nearby, thereby producing an amplification product. As this occurs, an increase in fluorescence occurs (see Appendix F and G attached hereto). In this system, a polymerase having 5'-3 'exonuclease activity is required.

Another embodiment utilizes a detection method for real-time amplification measurements that includes the use of a pair of amplification primers, one of which matches the random sequence of primer 2. Label one of these primers in the pair with a fluorescent group that fluoresces only when incorporated into the PCR product, as is the Lux ^™ primer known in the art (see Appendix H attached hereto). To do. In such embodiments, prior to incorporation, the sequence in the primer / probe binds to the complementary sequence in the primer / probe containing the fluorescent group and quenches the fluorescent group for incorporation into a double stranded product. Prior to this, the fluorescent group is quenched by the second structure. In another embodiment, primers 1 and 2 are generated only when primers 1 and 2 are ligated and amplified when hybridized to the amplified target sequence, and they fluoresce (Appendix) E or A), thus a FRET partner that allows the detection of the amplified target sequence. In a preferred embodiment, either the fluorescence detection is carried out beyond the the T _m for Tm or primer, P2 for primers P1, alternatively, go beyond the T _m of a both primers P1 and P2, were amplified Avoid background signal from possible hybridization of P1 and / or P2 to the target.

In another embodiment, the primer can be designed to exponentially amplify a target nucleic acid product that can be discerned by increasing or decreasing the melting temperature (T _m ), where it is amplified exponentially. The target sequence is stabilized as indicated by an increase in T _m , or destabilized as indicated by a decrease in T _m relative to the melting temperature of the linearly generated non-target product generated from the unligated primer It becomes. Variations in the random sequence or elsewhere in the primer can be used to generate such an exponentially amplified target nucleic acid sequence that can be distinguished from the linear product by the melting temperature.

  In another embodiment, probes specific for hybridizing across the ligation junction formed after ligation of the first and second primers can be used. Such probes can have a hairpin configuration at one end of the fluorescent reporter group and the other end of the fluorescence quenching group so that no fluorescence occurs if the probe does not bind across the linking junction. . By optimizing reaction conditions such as temperature and / or ionic strength, the hairpin is stabilized by binding across the ligation junction, where fluorescence occurs and the emission can be monitored to detect the amplified product. Like.

(Example 2 Single tube reaction system for identifying SNP)
One preferred embodiment of the present invention is the single tube reaction system shown in FIG. Similar to the embodiment shown in FIGS. 1 and 2 and discussed above in Example 1, a three primer system is utilized to identify the SNP of interest in the target sequence of the nucleic acid. Again, there are upstream and downstream primers that bind to the target nucleic acid in close proximity to the SNP of interest. The 3 ′ end of the upstream primer can be directly adjacent to the 5 ′ end of the downstream primer, or there is a gap of about 1 to 4 bases between the 3 ′ end of the upstream primer and the 5 ′ end of the downstream primer. possible. Either the 3 ′ end of the upstream primer or the 5 ′ end of the downstream primer can comprise a complement to the SNP of interest in the target nucleic acid.

  However, unlike the embodiments shown in FIGS. 1 and 2, the single tube reaction system provides simultaneous single tube identification and a distinction between one or more SNPs of interest in one or more target nucleic acid sequences of interest. Make it possible. This is accomplished by using unique sequences in each of the upstream and downstream primers (two linked) and the random sequence region of the general extension primer. As seen in FIG. 4A, the single tube reaction system includes a first upstream primer AB with a random sequence A that identifies the first SNP of interest in the first target nucleic acid segment, and a second Identifies the second SNP of interest in the target nucleic acid segment, has a second upstream primer FE with a random sequence F, and a random sequence D ′ complementary to the random sequence D present in the downstream primer CD A generic extension primer can be included, where C is common to both target nucleic acid segments.

  Upon successful identification and binding to a target nucleic acid having the SNP of interest, upstream primer AB and / or FE is ligated to downstream primer CD, and ligation products ABCD and / or Or F-E-C-D. If a gap exists between the 3 'end of the upstream primer and the 5' end of the downstream primer, the gap is first filled by polymerase activity and subsequently ligated to form a ligation product. The extension of both ligation products then takes place with the general extension primer D ', resulting in extended products A'-B'-C'-D' and F'-E'-C'-D '.

  The hybridizable probe A with fluorophore 1 and the hybridizable probe F with fluorophore 2 are then extended to the extended products A′-B′-C′-D ′ and F′-E, respectively. Hybridize to '-C'-D', followed by incorporation of each probe with that particular fluorescent tag into the amplified product (ABCD or FECD) And amplification occurs to trigger fluorescence of either fluorophore 1 or fluorophore 2 or both. In this way, one or more SNPs can be identified and distinguished in a single tube reaction system by monitoring the fluorescence signal of two (or more) fluorophores upon incorporation into the amplified product. .

  In another embodiment, an alternative single tube reaction system for identifying and distinguishing one or more SNPs in one or more target nucleic acid segments is shown in FIGS. 5A-5F. FIGS. 5A-5C are identical to FIGS. 4A-4C in that upstream primers AB and FE, downstream primer CD, and general extension primer D 'are present in a single tube reaction system. Again, the 3 ′ end of the upstream primer can contain the complement to the SNP of interest in the target nucleic acid, or the 5 ′ end of the downstream primer can contain the complement of the SNP of interest in the target nucleic acid, Upon binding to the target nucleic acid, the two primers can be adjacent or can have a gap of about 1 to 4 bases between the 3 ′ end of the upstream primer and the 5 ′ end of the downstream primer. Must be filled by the polymerase before ligation between the upstream and downstream primers can occur.

  However, as shown in FIG. 5D, an alternative single tube reaction system does not use hybridizable probes A and F with fluorophores 1 and 2 to amplify the target nucleic acid, but rather extended product A. Regular to amplify '-B'-C'-D' and F'-E'-C'-D 'to the amplified target nucleic acid product ABCD or FECD Primers A and F are used. Such a system would be advantageous when specific target nucleic acids are not effectively amplified with a hybridizable probe having a bulky fluorophore attached to them. In this alternative single tube reaction system, the amplified target nucleic acid sequences dissolve at a temperature where they are shorter and lower than the annealing temperature of primers A and F (or fluorescent probes A and F in FIG. 4). Detected after amplification by hybridizable probes hyb-A and hyb-F with additional fluorescent tags that differ from the normal primers A and F in that they have secondary structure. This is due to the extended products A'-B'-C'-D 'and F'-E'-C'-D' to the target nucleic acid products A-B-C-D or F-E-C-D. Allows ineffective competition between the hyb-A and hyb-F probes and the normal primers A and F, but the competing reaction is sufficient to measure the fluorescence of fluorophores 1 and 2 Allows to occur, thereby allowing detection and quantification of the amplified target nucleic acid product.

  The use of a general extension primer such as D ′ that is complementary to sequence D in segment CD common to both target nucleic acid segments is described above and in the single tube reaction system illustrated in FIGS. Although convenient, it is not required. It is believed that a single tube reaction system can be adapted to produce ligation products with AB and FE using more than one extended primer simultaneously. The selectivity of the first primer AB for the first SNP and the second primer EF for the second SNP ensures selective ligation, and the CX product to be ligated even with additional primers Is used to generate

  Upon successful identification and binding of a target nucleic acid having the SNP of interest, upstream primers AB and / or FE are ligated to downstream primers CG and CH, respectively, and ligation product A- This produces BCG and / or FECH. If a gap exists between the 3 'end of the upstream primer and the 5' end of the downstream primer, the gap is first filled by polymerase activity and subsequently ligated to form a ligated product. Extension of both ligation products then takes place with extension primers G ′ and H ′, resulting in extended products A′-B′-C′-G ′ and F′-E′-C′-H ′. be able to.

  As described above, a) monitor the fluorescence signal of two (or more) fluorophores upon incorporation into the amplified product, or b) hybridizable probes hyb-A and hyb-F with additional fluorescent tags Can be used to identify and distinguish one or more SNPs in a single tube reaction system by detecting the fluorescent signal after amplification.

(Example 3 Nanoliter sampling array)
Another embodiment of the invention includes a nanoliter sampling array. Any currently available array in the prior art may be used, but US Provisional Application Nos. 60 / 518,240 and November 2004 filed Nov. 7, 2003, both incorporated herein by reference. A particular application array similar to that described in US application Ser. No. 10 / 984,027 filed on the 8th is one preferred array. In this particular embodiment, the array includes a first platen having at least one hydrophobic surface and having a dense microfluidic array of hydrophilic through holes. A target nucleic acid sequence is selected and an array is prepared, wherein each through hole in the array is at its 3 ′ end that is substantially complementary to the first segment at the first end of the nucleic acid target sequence. A first primer having at least a portion, and at least a second primer having at least a portion of its 5 ′ end that is substantially complementary to a second segment at the second end of the nucleic acid target sequence; The 5 ′ end of the second primer is adjacent to the 3 ′ end of the first primer upon binding to a potential nucleic acid target sequence. FIG. 4 shows such an array as known in the prior art. The array chip 10 typically has a thickness of 0.1 mm to 10 mm or more; for example, a thickness of 0.3 to 1.52 mm, usually 0.5 mm. A typical volume for the sample through hole 12 can be 0.1 picoliter to 1 microliter, and a typical volume is in the range of 0.2 to 100 nanoliters, for example, about 35 nanoliters. The sample through hole 12 can be loaded using the capillary action or surface tension of the liquid sample. For typical chip dimensions, the capillary force is strong enough to hold the liquid in place. The tip loaded with the sample solution can be rocked in the air and can even be centrifuged at moderate speed without displacing the sample.

  To enhance the suction power of the sample through hole 12, the target area of the receptacle inner wall 42 can have a hydrophilic surface that attracts the liquid sample. Alternatively, the sample through hole 12 can include a porous hydrophilic material that attracts the liquid sample. In some embodiments, sample through-holes in the array can be coated with a biocompatible material such as polyethylene glycol, and the primer can be passed through the through-hole by drying the primer after application to the through-hole. It can be deposited on, in or under the surface biocompatible material. In order to prevent cross-contamination, the outer planar surface 14 of the chip 10 and the layer of material 40 around the opening of the sample through hole 12 can be of a hydrophobic material. Thus, each sample through hole 12 has an internal hydrophilic region bonded to either end by a hydrophobic region.

  The through hole design of the sample through hole 12 avoids the trapped air problem inherent in other microplate structures. This approach, together with hydrophobic and hydrophilic patterning, allows self-weighing loading of the sample through hole 12. The self-loading function helps manufacture the array with pre-loaded reagents and helps the array fill itself when contacted with aqueous sample material.

Example 3 Method for Identifying SNPs in a Nucleic Acid Target Sequence
Yet another embodiment is a method of identifying a single nucleotide polymorphism (SNP) in a target sequence of a nucleic acid. Identify the target sequence of the nucleic acid, so that the two primers P1 and P2 are designed to be in close proximity to the SNP located internally on one strand of the target nucleic acid sequence and are linked by a thermally stable ligase Primers are prepared according to standard methods as designed. Primers P1 and P2 are further designed such that the base complementary to the SNP in the target sequence is either at the 3 ′ end of P1 or at the 5 ′ end of P2. In this special method, a nanoliter sampling array is used. The method includes providing a first platen having a high density microfluidic array of through holes, wherein each through hole of the array is substantially relative to a first segment at a first end of the target sequence. A first primer having at least a portion of its 3 ′ end that is substantially complementary, and at least a portion of its 5 ′ end that is substantially complementary to a second segment at the second end of the target sequence A second primer having: Upon binding to the target sequence, the 5 ′ end of the second primer is adjacent to the 3 ′ end of the first primer.

  The method further includes introducing a sample containing a target nucleic acid sequence having an internal SNP into the array and then introducing a reagent into a through hole in the array, wherein the reagent is a linked primer. A third primer having a random sequence capable of amplifying the P1-P2 product, a thermostable polymerase, a thermostable ligase, and at least four different nucleoside triphosphates. Further steps in the method include subjecting the array to thermal cycling with primers, target nucleic acids, and reagents, and then detecting the resulting amplified target nucleic acid sequence. If desired, the thermostable polymerase can lack 5'-3 'exonuclease activity, or it can lack 3'-5' exonuclease activity, or it can be 5'-3 'and 3'-5. 'Can lack both exonuclease activities.

  The detection step can also include the use of a dye specific for binding to double stranded DNA or RNA that fluoresces upon binding to the amplified target sequence. Suitable dyes are SYBR® Green I, SYBR® Green II, YOYO®-1, TOTO®-1, POPO®-3, EtBr, and fluorescence. It includes any other dye that can provide insensitive detection of the amplified target sequence.

  Alternatively, detection can occur through the addition of a probe specific for hybridization across the ligation junction of ligated P1-P2 primer products, where such a probe is like a fluorescent quencher. Fluorescent groups and fluorescent modifying groups.

  In another alternative embodiment, detection can include the use of a probe comprising a fluorescent group and a fluorescent modifying group, such as a fluorescent quencher specific for hybridizing to a region of the target sequence. . In this particular embodiment, the fluorescent modifying group is cleaved upon extension of the probe, and the fluorescent group thus fluoresces, allowing detection of the amplified product.

  Further embodiments of the invention include a kit for use in identification of an amplified target nucleic acid sequence, wherein the kit has one hydrophobic surface and a dense microfluidic array of hydrophilic through-holes Provide platen. In one particular kit, each through-hole has a first primer having at least a portion of its 3 ′ end that is substantially complementary to the first segment at the first end of the potential nucleic acid target sequence. A second primer having at least a portion of its 5 ′ end that is substantially complementary to a second segment at the second end of the potential nucleic acid target sequence, the 5 ′ end of the second primer being A reagent platen adjacent to the 3 ′ end of the first primer upon binding to a potential nucleic acid target sequence, and having a high-density microfluidic array of through holes, each through hole being random at the 3 ′ end of the second primer A third primer that is substantially complementary to the sequence segment and to a substantially similar sequence at the 5 ′ end of the first primer. Includes, it contains at least four different nucleotide bases, a thermostable ligase and fluorescent dyes. In this particular embodiment, the reagent platen has a structural geometry corresponding to the sample platen that allows delivery of the reagent components and the target nucleic acid sample to a primer in the sample platen. In some embodiments of the kit, the primer may be deposited in or under a biocompatible material, such as a wax-like coating in the through hole, by drying the primer after it is applied to the through hole. Here, the biocompatible material can include, for example, a polyethylene glycol (PEG) material. In order to perform selective ligation and amplification for identification of the amplified target nucleic acid sequence, the user simply selects a sample containing the target nucleic acid, a thermostable polymerase, and optionally a buffer provided with the kit. Will add to the through hole.

FIG. 1-A shows a double-stranded target nucleic acid sequence with a single nucleotide polymorphism (SNP). FIG. 1-B1 shows the 3′-5 ′ target strand denatured with primers 1 and 2 hybridized adjacent to the SNP, the base complementary to the SNP located at the 3 ′ end of primer 1, and The random sequence (RS) of primer 3 hybridized to the 3 ′ end of the linked P1-P2 product is shown. FIG. 1-B2 shows the 3′-5 ′ target strand denatured with primers 1 and 2 hybridized adjacent to the SNP, the base complementary to the SNP located at the 5 ′ end of primer 2, and The random sequence (RS) of primer 3 hybridized to the 3 ′ end of the linked P1-P2 product is shown. FIG. 1-C shows the denatured 5'-3 'target nucleic acid strand extended by the unlinked primer P1. FIG. 2-A shows a double-stranded target nucleic acid sequence with a single nucleotide polymorphism (SNP). FIG. 2-B shows primers P1 and P2 hybridized to the denatured target strand (3′-5 ′ strand) of the nucleic acid, where the base complementary to the SNP in the target strand is P1 3 ′. Present at the ends, each of the primers P1 and P2 includes random sequences at their 5 ′ and 3 ′ ends, respectively. FIG. 2-C shows the ligated P1-P2 product that is amplified by primer P3 to yield the P3-amplified product. FIG. 2-D shows a P3-amplification product that is amplified by primer P3 to yield a P2-amplification product (3'-5 '). Figures 2-E1 and 2-E2 show the exponential amplification of P3-amplification product (5'-3 ') and P3-amplification product (3'-5'), respectively. FIG. 3 shows the dye SYBR® Green I binding to a double-stranded amplified target nucleic acid and the fluorescence emission cartoon. FIG. 4-A shows a target having a SNP of interest in a single tube reaction system for discriminating between upstream primer AB, downstream primer CD, and one or more SNPs in one or more target sequences of nucleic acids. A general extension primer D ′ with nucleic acids is shown, the single tube reaction system also includes an upstream primer FE and a second nucleic acid target with a second SNP of interest. FIG. 4-B shows the ligation of upstream primer AB and downstream primer CD when a successful match occurs with the first SNP of interest in the first target sequence of the nucleic acid, and is identical. Also shown is ligation of the upstream primer FE and the downstream primer CD when a successful match occurs with the second SNP of interest in the second target sequence of the nucleic acid present in the tube. FIG. 4-C shows the extension of the ligation products ABCD and FECD by the general extension primer D '. FIG. 4-D shows hybridization of hybridizable probe A with fluorescent tag 1 to extended product A′-B′-C′-D ′, and hybridizable probe with fluorescent tag 2 Shown is the hybridization of F to the extended product F′-E′-C′-D ′. FIG. 4-E shows the uptake and amplification of the first target nucleic acid with the first SNP of interest by hybridizable probe A that triggers fluorescence of fluorophore 1 in the first amplified product, and FIG. 6 shows the uptake and amplification of a second target nucleic acid with a second SNP of interest by hybridizable probe F that triggers fluorescence of fluorophore 2 in two amplified products. FIG. 5A shows a target nucleic acid with upstream primer AB, downstream primer CD, and SMP of interest in a single tube reaction system for discriminating between one or more SNPs in one or more target sequences of the nucleic acid. Wherein the single tube reaction system is an upstream primer FE, and a second nucleic acid target having the SNP of interest in an alternative embodiment of the single tube reaction system of FIG. Including. FIG. 5B shows the ligation of upstream primer AB and downstream primer CD when a successful match occurs with the first SNP of interest in the first target sequence of the nucleic acid, and in the same tube. Figure 8 shows the ligation of upstream primer FE and downstream primer CD when a successful match occurs with the second SNP of interest in the second target sequence of the nucleic acid present. FIG. 5C shows the extension of the ligation products ABCD and FECD by the general extension primer D '. FIG. 5D shows hybridization of primer A without fluorescent tag to extended product A′-B′-C′-D ′, and extended product F′-E ′ of primer F without fluorescent tag. Hybridization to -C'-D'- is shown. FIG. 5E shows the amplification of the first target nucleic acid with the first SNP of interest by primer A to produce the first amplified product, and the second amplified product to produce the second amplified product, Amplification of a second target nucleic acid with a second SNP of interest by primer F is shown. FIG. 5F shows a competition reaction for the amplification reaction in FIG. 5E, where the uptake and low efficiency generation of the first target nucleic acid with the first SNP of interest is performed by the hybridizable probe A, Triggering the fluorescence of fluorophore 1 in the product of thereby allowing the detection of the first amplified target nucleic acid and the uptake and low efficiency of the second target nucleic acid with the second SNP of interest here Production is performed by the hybridizable probe F, triggering the fluorescence of the fluorophore 2 in the second product, thereby allowing the simultaneous detection of the second amplified target nucleic acid. FIG. 6 shows a typical high density sample array of through holes according to the prior art.

Claims (86)

An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, said assay comprising a first of said target sequence and each of said target sequences A ligable first primer and a second primer having at least a portion that is substantially complementary to the first segment and the second segment, and substantially to the random sequence segment of the first primer and the second primer A selective ligation and amplification method of the type using a controlled temperature reaction mixture comprising a third primer that is complementary to
An improved assay comprising homogeneously detecting the amplified target sequence using a dye specific for binding to double-stranded (ds) DNA that fluoresces upon binding to the target sequence. The improved assay of claim 1, wherein a nucleotide complementary to the SNP of the target sequence is present at the 5 'end of the second primer. The improved assay of claim 1, wherein the dye comprises SYBR® Green. The assay is
Use the first and second primers at a concentration such that the ligated product yields a detectable exponentially amplified labeled sequence that exceeds the linearly amplified unlinked primer product The improved assay of claim 1 further comprising the step of: The assay is
Using a plurality of first and second primers designed to produce amplified target sequences having different melting curves;
The improved assay of claim 1, further comprising the step of identifying individual amplified label sequences by differences in melting temperature ( _Tm ). The improved assay of claim 1, wherein the first primer and the second primer comprise degenerate base-pairing positions that allow hybridization to a variable region in a target sequence adjacent to the SNP. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, said assay comprising a first of said target sequence and each of said target sequences A ligable first primer and a second primer having at least a portion that is substantially complementary to the first segment and the second segment, and a random sequence segment of the first primer and the second primer A type of selective ligation and amplification method using a temperature-controllable reaction mixture comprising a third primer that is substantially complementary, wherein the improvement is:
Target sequence amplified using a probe specific for hybridizing across the ligation junction formed between the first primer and the second primer after binding to the target sequence An improved assay, wherein the probe specific for hybridizing across the ligation junction comprises a molecular beacon. 8. The improved assay of claim 7, wherein the probe specific for hybridizing across the ligation junction has a fluorescent group and a fluorescent modifying group. 9. The fluorescent group of claim 8, wherein the fluorescent group is quenched when the probe is not bound across the linking junction and the fluorescent group fluoresces when the probe binds across the linking junction. Improved assay. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, said assay comprising a first of said target sequence and each of said target sequences A ligable first primer and a second primer having at least a portion substantially complementary to the segment and the second segment, and a random sequence segment of the first primer and the second primer A selective ligation and amplification method of the type using a temperature-controllable reaction mixture comprising a third primer that is substantially complementary.
Detecting the amplified target sequence using a probe specific for hybridizing to a region of the target sequence, wherein the probe comprises a fluorescent group and a fluorescent modifying group. Assay. 11. The improved assay of claim 10, wherein upon extension of the probe, the fluorescent modifying group is excised and the fluorescent group fluoresces. 11. An improved assay according to claim 7 or 10, wherein the fluorescent group is quenched prior to incorporation into a double stranded product and dequenched after incorporation into a double stranded product. The double-stranded product, such that the fluorescent group quenches the fluorescent group by binding to the complementary sequence in the probe containing the fluorescent group prior to incorporation into the double-stranded product. 13. The improved assay of claim 12, which is quenched by secondary structure prior to incorporation into. A nanoliter sampling assay comprising:
a) comprising a first platen having at least one hydrophobic surface and having a dense microfluidic array of hydrophilic through-holes;
Here, each through hole is
i) a first primer having at least a portion of the 3 ′ end substantially complementary to the first segment at a first end of the potential nucleic acid target sequence; and ii) of the potential nucleic acid target sequence A second primer having, at the second end, at least a portion of the 5 'end that is substantially complementary to the second segment;
Including
The nanoliter sampling array, wherein the first primer and the second primer are ligable upon binding to the potential nucleic acid target sequence. The nanoliter sampling assay of claim 14, further comprising:
A second platen having at least one hydrophobic surface and having a dense microfluidic array of hydrophilic through holes;
Here, the first platen and the second platen are fixedly connected so that the respective through holes are aligned, a nanoliter sampling array. 15. The nanoliter sampling array of claim 14, wherein at least one pair of aligned through holes comprises a first reagent for a first assay process and a second reagent for a second assay process. The array of claim 16, wherein one of the assay processes is PCR amplification. The array of claim 16, wherein one of the assay processes is detection of an amplified target nucleic acid sequence having a SNP. 19. The detection of the amplified target nucleic acid sequence comprises using a dye specific for binding to double stranded (ds) DNA that fluoresces upon binding to the target sequence. The described array. 19. An array according to claim 18, wherein detection of amplified target nucleic acid sequences comprises identifying individual amplified target sequences by differences in melting temperature ( _Tm ). Detection of amplified target nucleic acid sequence is specific for hybridizing across the ligation junction formed between the first and second primers after binding to the target sequence 19. The array of claim 18, comprising using a simple probe, wherein the probe has a fluorescent group and a fluorescent modifying group. Detection of the amplified target nucleic acid sequence includes using a probe containing a fluorescent group and a fluorescent modifying group specific for hybridizing to a region of the target sequence, wherein the probe comprises: 19. The array of claim 18, wherein upon extension, the fluorescent modifying group is excised and the fluorescent group fluoresces. 23. The array of claim 22, wherein the fluorescent group is quenched prior to incorporation into a double stranded product and dequenched after incorporation into a double stranded product. The fluorescent group is double-stranded so that the sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group and quenches the fluorescent group prior to incorporation into the double-stranded product. 24. The array of claim 23, which is quenched by secondary structure prior to incorporation into the product. 25. A nanoliter sampling array according to any one of claims 14 to 24, wherein the primer is attached by drying onto, within, or under a sample through-hole coating, the coating comprising a biocompatible material. A method for identifying a SNP in a target sequence of a nucleic acid, the method comprising:
Providing a first sample platen having a dense microfluidic array of through-holes, each through-hole having at least a portion that is substantially complementary to a first segment of the target sequence. A second primer having at least a portion substantially complementary to a second segment of the target sequence, and a random sequence segment of the first primer and the second primer. A third primer that is complementary to the second primer, the 5 ′ end of the second primer being ligable to the 3 ′ end of the first primer after binding of the nucleic acid target sequence;
Introducing a sample containing a target sequence of a nucleic acid having a target SNP into a through hole in the array;
Introducing a reagent into a through hole in the array, the reagent comprising a reagent for performing amplification, a reagent for performing ligation, and at least four different nucleotide bases;
Ligating the first primer and the second primer to generate a ligation product;
Amplifying the ligation product and target sequence;
Detecting the amplified target sequence. 27. Identifying a SNP in a target sequence of a nucleic acid according to claim 26, wherein the steps of performing ligation and performing amplification include adding ligase and polymerase followed by subjecting the array to controlled temperature conditions. Method. 27. The method of claim 26, wherein the detecting comprises using a dye specific for binding to double stranded (ds) DNA that fluoresces upon binding to the target sequence. 27. The method of claim 26, wherein detecting comprises identifying individual amplified target sequences by differences in melting temperature ( _Tm ). The detecting step uses a probe specific for hybridizing across the ligation junction formed between the first primer and the second primer after binding to the target sequence. 27. The method of claim 26, comprising a step wherein the probe has a fluorescent group and a fluorescent modifying group. Detecting comprises using a probe containing a fluorescent group and a fluorescent modifying group specific for hybridizing to a region of the target sequence, wherein upon extension of the probe, 27. The method of claim 26, wherein the fluorescent modifying group is cleaved and the fluorescent group fluoresces. 32. The method of claim 30, wherein the fluorescent group is quenched prior to incorporation into a double stranded product and dequenched after incorporation into a double stranded product. The fluorescent group is double-stranded so that the sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group and quenches the fluorescent group prior to incorporation into the double-stranded product. 33. The method of claim 32, wherein the method is quenched by secondary structure prior to incorporation into the product. A kit for use in identification of an amplified target nucleic acid sequence, the kit comprising: a) and b):
a) a sample platen having one hydrophobic surface and having a dense microfluidic array of hydrophilic through-holes,
Here, the through hole of each sample platen is at least
i) a first primer having at least a portion substantially complementary to a first segment of a potential nucleic acid target sequence;
ii) comprising a second primer having at least a portion substantially complementary to a second segment of the potential nucleic acid target sequence, wherein the first primer and the second primer are the potential nucleic acid target sequence A sample platen that is connectable upon binding to
b) a reagent platen having a high-density microfluidic array of through-holes,
The through hole of each reagent platen is at least
i) a third primer that is substantially complementary to the random sequence segments of the first primer and the second primer;
ii) at least four different nucleotide bases iii) a reagent for performing the ligation; and iv) a reagent platen comprising a fluorescent dye,
With
The kit, wherein the reagent platen has a structural geometry corresponding to the sample platen that allows delivery of reagent components and target nucleic acid sample to a primer in the sample platen. 35. A kit for use in the identification of amplified target nucleic acid sequences according to claim 34, which also includes a PCR compatible buffer. The fluorescent dye is SYBR (registered trademark) Green I, SYBR (registered trademark) Green II, YOYO (registered trademark) -1, TOTO (registered trademark) -1, POPO (registered trademark) -3, or ethidium bromide. 35. The kit of claim 34, comprising. 37. A kit according to any of claims 34 to 36, wherein the primer is attached by drying onto, within, or under a sample through-hole coating, the coating comprising a biocompatible material. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, said assay comprising a first of said target sequence and each of said target sequences A ligable first primer and a second primer having at least a portion substantially complementary to the segment and the second segment, and a random sequence segment of the first primer and the second primer A selective ligation and amplification method of the type using a controlled temperature reaction mixture comprising a third primer that is substantially complementary.
Detecting one or more amplified target sequences in a single tube reaction system using one or more probes specific for hybridizing to a region of one or more target nucleic acid sequences, comprising: An improved assay wherein each of the one or more probes contains a distinct fluorescent group and a fluorescent modifying group, and wherein hybridization of the one or more probes results in fluorescence of the distinct fluorescent group. 40. The improved assay of claim 38, wherein upon extension of the probe, the fluorescent modifying group is cleaved and the fluorescent group fluoresces. 40. The improved assay of claim 38, wherein the fluorescent group is quenched prior to incorporation into a double stranded product and dequenched after incorporation into a double stranded product. The fluorescent group is double-stranded so that the sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group and quenches the fluorescent group prior to incorporation into the double-stranded product. 41. The improved assay of claim 40, wherein the improved assay is quenched by secondary structure prior to incorporation into the product. 40. The improved assay of claim 38, wherein the one or more target nucleic acid sequences are two, each having a distinct SNP of interest. 43. The one or more hybridizable probes are two, each having a unique sequence that hybridizes to and amplifies each of a separate fluorophore and two target nucleic acid sequences. The improved assay described. 40. The improved assay of claim 38, wherein the one or more target nucleic acid sequences are three, each having a distinct SNP of interest. 45. The one or more hybridizable probes are three, each having a unique sequence that hybridizes to and amplifies each of a separate fluorophore and three target nucleic acid sequences. The improved assay described. 40. The improved assay of claim 38, wherein the one or more target nucleic acid sequences are four, each having a distinct SNP of interest. The said one or more hybridizable probes are four, each having a unique sequence that hybridizes to and amplifies each of a separate fluorophore and four target nucleic acid sequences. Improved assay. An improved assay of the type for amplifying a specific target nucleic acid sequence, wherein the target sequence comprises an internal SNP of interest, said assay comprising a first of said target sequence and each of said target sequences A ligable first primer and a second primer having at least a portion substantially complementary to the segment and the second segment, and a random sequence segment of the first primer and the second primer A selective ligation and amplification method of the type using a controlled temperature reaction mixture comprising a third primer that is substantially complementary to, wherein the improvement comprises:
Detecting one or more amplified target sequences in a single tube reaction system using one or more fourth primers, each having a fluorescent group and a fluorescent modifying group, and each having one or more Complementary to a unique region of the template linked to the target nucleic acid sequence of the template, wherein the distinction during the incorporation of the fourth primer into the one or more target nucleic acid sequences and amplification thereof The improved assay wherein the fluorescence of the fluorescent group occurs such that detection of one or more amplified target nucleic acid sequences is obtained in a single tube reaction system. 49. The improved assay of any of claims 1, 7, 10, 26, or 48, further comprising using a polymerase that lacks 5'-3 'exonuclease activity. 49. The improved assay of any of claims 1, 7, 10, 26, or 48, further comprising using a polymerase that lacks 3'-5 'exonuclease activity. 49. The improved assay of any of claims 1, 7, 10, 26 or 48, further comprising using a polymerase that lacks 5'-3 'exonuclease activity and 3'-5' exonuclease activity. 49. The separate fluorescent group comprises Redmond Red ^™ , Yakima Yellow ^™ , and the fluorescent modifying group comprises an Eclipse ^™ non-fluorescent quencher, dabsyl, or other fluorescence quenching molecule. An improved assay according to any of the above. 49. The improved assay of claim 48, wherein the one or more target nucleic acid sequences are two, each having a distinct SNP of interest. 54. The one or more fourth primers are two, each having a unique fluorophore and a unique sequence that is incorporated into and amplifies each of two target nucleic acid sequences. Improved assay. 49. The improved assay of claim 48, wherein the one or more target nucleic acid sequences are three, each having a distinct SNP of interest. 56. The improvement of claim 55, wherein the one or more fourth primers are three, each having a unique fluorophore and a unique sequence that is incorporated into and amplifies each of the three target nucleic acid sequences. Assay. 49. The improved assay of claim 48, wherein the one or more target nucleic acid sequences are four, each having a distinct SNP of interest. 58. The one or more fourth primers are four, each having a unique sequence that is incorporated into and amplifies each of a separate fluorophore and four target nucleic acid sequences. Improved assay. A nanoliter sampling assay,
a) a first platen having at least one hydrophobic surface and having a dense microfluidic array of hydrophilic through-holes;
Here, the through hole of each first platen is at least
i) one or more first primers each having at least a portion that is substantially complementary to a first segment of the one or more target nucleic acid sequences; and ii) the first of the one or more target nucleic acid sequences A second primer having a portion that is substantially complementary to two segments, wherein the first primer and the second primer are ligable upon binding to the one or more target nucleic acid sequences. A nanoliter sampling array. 60. The nanoliter sampling array of claim 59, wherein
A second platen having at least one hydrophobic surface and having a dense microfluidic array of hydrophilic second platen through-holes, wherein the first platen and the second platen Is a nanoliter sampling array where each through-hole is fixed and connected so that it is aligned. 60. The nanoliter sampling of claim 59, wherein the at least one pair of aligned through holes comprises at least a first reagent for the first assay process and a second reagent for the second assay process. array. 62. The array of claim 61, wherein one of the assay processes is PCR amplification. 62. The array of claim 61, wherein one of the assay processes is the detection of one or more amplified target nucleic acid sequences each having a SNP. Detection of the one or more amplified target nucleic acid sequences comprises using one or more probes specific for hybridizing to each region of the one or more target sequences, each probe separately Each of the separate fluorescent modifying groups is cleaved upon extension of the one or more probes to one or more amplified target nucleic acid sequences, and 64. The array of claim 63, wherein the separate fluorescent group fluoresces. Detection of the one or more amplified target nucleic acid sequences comprises using one or more probes specific for hybridizing to each region of the one or more target sequences, each probe separately 64. The fluorescent group of claim 63, wherein the fluorescent group is quenched prior to incorporation into a double-stranded product and dequenched after incorporation into a double-stranded product. Array. The fluorescent group is double stranded such that the sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group and quenches the fluorescent group prior to incorporation into a double stranded product. 66. The array of claim 65, which is quenched by secondary structure prior to incorporation into the product. 67. A nanoliter sampling array according to any one of claims 59 to 66, wherein the primer is bonded by drying onto, within or under a sample through-hole coating, the coating comprising a biocompatible material. A method for identifying one or more SNPs in one or more target nucleic acid sequences, the method comprising:
Providing a first sample platen having a high-density microfluidic array of through-holes, each through-hole in each sample platen to at least one or more first primers and a second segment of the target sequence A second primer having at least a portion that is substantially complementary to each other and a third primer that is substantially complementary to a random sequence segment of the second primer, each first primer Has at least a portion that is substantially complementary to a first segment of the one or more target nucleic acid sequences, and the 5 ′ end of the second primer is after binding to the one or more target nucleic acid sequences. A step capable of ligation to the 3 ′ end of the first primer;
Introducing a sample containing nucleic acids of one or more target sequences each having a target SNP into a through hole of a sample platen in the array;
Introducing a reagent into a through-hole of a sample platen in the array, the reagent comprising a reagent for performing amplification, a reagent for performing ligation, and at least four different nucleotide bases;
Ligating the first primer and the second primer to produce a ligation product;
Performing amplification of the ligation product and one or more target sequences; and detecting one or more amplified target sequences. 69. Identifying a SNP in a target sequence of a nucleic acid according to claim 68 wherein the steps of performing ligation and performing amplification include adding ligase and polymerase followed by subjecting the array to controlled temperature conditions. Method. Before introducing the reagent into the through hole of the sample platen in the array,
Further comprising introducing a sample containing one or more probes specific for hybridizing to a region of one or more target nucleic acid sequences, and amplifying the one or more target sequences, wherein 69. The method of identifying one or more SNPs of claim 68, wherein the one or more probes each contain a separate fluorescent group and fluorescent modifying group. 71. Upon extension of the one or more probes to one or more amplified target nucleic acid sequences, each of the separate fluorescent modifying groups is excised and the separate fluorescent groups fluoresce. A method for identifying one or more SNPs. 72. The method of identifying one or more SNPs of claim 70, wherein the fluorescent group is quenched prior to incorporation into a double stranded product and dequenched after incorporation into a double stranded product. The fluorescent group is double-stranded so that the sequence in the probe binds to a complementary sequence in the probe containing the fluorescent group and quenches the fluorescent group prior to incorporation into the double-stranded product. 75. The method for identifying one or more SNPs of claim 72, wherein the one or more SNPs are quenched by secondary structure prior to incorporation into the product. The step of identifying one or more SNPs in one or more target nucleic acid sequences comprises monitoring different fluorescence of the one or more distinct fluorescent groups incorporated into one or more amplified target nucleic acid sequences. 71. The method according to 71. 69. A method of identifying one or more SNPs in one or more target sequences of a nucleic acid of claim 68, wherein the polymerase lacks 5'-3 'exonuclease activity. 69. A method of identifying one or more SNPs in a target sequence of a nucleic acid according to claim 68, further comprising using a polymerase that lacks 3'-5 'exonuclease activity. 69. A method of identifying one or more SNPs in one or more target nucleic acid sequences of claim 68, further comprising using a polymerase lacking 5'-3 'exonuclease activity and 3'-5' exonuclease activity. . 72. The method of claim 70, wherein the one or more target nucleic acid sequences are two, each having a distinct SNP of interest. 79. The one or more hybridizable probes are two, each having a unique fluorophore and a unique sequence that hybridizes to and amplifies each of two target nucleic acid sequences. the method of. 71. The method of claim 70, wherein the one or more target nucleic acid sequences are three, each having a distinct SNP of interest, 81. The one or more hybridizable probes is three, each having a unique fluorophore and a unique sequence that hybridizes to and amplifies each of the three target nucleic acid sequences. the method of. 72. The method of claim 70, wherein the one or more target nucleic acid sequences are four, each having a distinct SNP of interest. 83. The four or more hybridizable probes are each four, each having a unique fluorophore and a unique sequence that hybridizes to and amplifies each of the four target nucleic acid sequences. the method of. A kit for use in the identification of one or more amplified target nucleic acid sequences, the kit comprising: a) and b):
a) a sample platen having one hydrophobic surface and having a dense microfluidic array of hydrophilic through-holes,
Here, the through hole of each sample platen is at least
i) one or more first primers each having at least a portion substantially complementary to a first segment of the one or more target nucleic acid sequences;
ii) containing a second primer having at least a portion substantially complementary to a second segment of the one or more target nucleic acid sequences, wherein the 3 ′ end of the one or more first primers comprises the A sample platen that can be ligated to the 5 ′ end of the second primer after binding to one or more target nucleic acid sequences;
b) a reagent platen having a high-density microfluidic array of through-holes,
The through hole of each reagent platen is at least
i) a third primer that is substantially complementary to the random sequence segment of the second primer;
ii) one or more probes specific for hybridizing to a region of one or more target nucleic acid sequences and amplifying the one or more target sequences, wherein the one or more probes are each One or more probes containing separate fluorescent groups and fluorescent modifying groups;
iii) four different nucleotide bases;
iv) ligase;
A reagent platen, the reagent platen having a structural geometry corresponding to the sample platen that allows delivery of the components of the reagent and a target nucleic acid sample to a primer in the sample platen . 85. A kit for use in the identification of amplified target nucleic acid sequences according to claim 84, which also includes a PCR compatible buffer. 86. A kit according to claim 84 or 85, wherein the primer is bound by drying onto, within or under a sample through-hole coating, the coating comprising a biocompatible material.

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