CN1944676A - Full genome rolling circle amplification and its product fixing method - Google Patents
Full genome rolling circle amplification and its product fixing method Download PDFInfo
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- CN1944676A CN1944676A CN 200610096842 CN200610096842A CN1944676A CN 1944676 A CN1944676 A CN 1944676A CN 200610096842 CN200610096842 CN 200610096842 CN 200610096842 A CN200610096842 A CN 200610096842A CN 1944676 A CN1944676 A CN 1944676A
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Abstract
全基因组滚环扩增及其产物的固定方法实现了全基因组信息的同时获取，大大节约了模板制备时间；同时由于全基因组的信息在片段化后同时以单分子形式扩增放大，既保持了片断化后的单分子数，又提高了使用检测的灵敏度。基因组DNA经酶消化或者超声破碎成较短的片段，片段化的DNA在酶的作用下连接连接子，并连接成环，以该环分子为模板进行滚环扩增，得到滚环扩增的基因组片段化核酸序列；将稀释的滚环扩增的基因组片段化核酸序列通过化学方法固定在一块硬质材料载体表面，使得每个基因组片段化核酸序列在该载体表面占有一个独立的位置坐标而与其它序列分开固定，从而实现全基因组序列的固定，构成全基因组DNA芯片。
Whole-genome rolling circle amplification and its product immobilization method realize the simultaneous acquisition of whole-genome information, which greatly saves template preparation time; The number of single molecules after fragmentation improves the sensitivity of detection. Genomic DNA is digested by enzymes or ultrasonically broken into shorter fragments, and the fragmented DNA is connected to linkers under the action of enzymes, and connected into a circle, and the circle molecule is used as a template for rolling circle amplification to obtain rolling circle amplification. Genome fragmented nucleic acid sequence; the diluted rolling circle amplified genomic fragmented nucleic acid sequence is chemically immobilized on the surface of a hard material carrier, so that each genome fragmented nucleic acid sequence occupies an independent position coordinate on the surface of the carrier. It is fixed separately from other sequences, so as to realize the fixation of the whole genome sequence and constitute a whole genome DNA chip.
Description
Technical field
The present invention relates to the process for fixation of a kind of whole genome amplification and product thereof, belong to the technical field that genome detects in the biomedicine.
Background technology
Along with going deep into of genome research, from the difference of gene level understanding life, disease takes place, the rule of development, and the interaction of medicine and life entity will become possibility.Though the factor that causes disease to take place is numerous, transgenation (single nucleotide polymorphism, methylate etc.) is widely regarded as an important internal factor.Aspect fundamental research, gene mutation site helps genomic accurate location, the genetic development of study of disease gene, clone's Disease-causing gene; In application facet, directly seek the susceptibility gene mutation site of disease: most of complex diseases, as cancer, diabetes, cardiovascular disorder, dysthymia disorders, asthma etc., be audient's polygene and environmental factor acting in conjunction, by identifying on a large scale for mutator gene type in the genome sample of a large amount of a certain specified diseases and detecting, can obtain about with the information of this disease related gene type.What is more important by the gene mutation site of examination medicaments insensitive, provides foundation for realizing personalized medicine from now on.By finding the gene mutation site of disease susceptibility, can make people's preventing disease early, reach the purpose of the generation of avoiding disease.Therefore, after the human genome sketch had just been completed, gene mutation site became one of main task of the Human Genome Project immediately in the searching human genome.For example, ten big pharmacy giant companies and Wellcome Trust have constituted jointly the SNP cooperative groups in the world, have started the plan of seeking the SNP site in the whole genome scope.China genome center, south and the north (China is big) gene center has also been carried out the screening of the SNP of Chinese nation gene locus.At present, location and certification work have been finished in most human SNP gene polymorphics site (about 3,000,000), and announce on the net.Yet, determine that the gene mutation site in the human genome only is the first step that people studied and utilized gene mutation site, seek gene mutation site and human phenotype, particularly the dependency with human diseases is only people's concern and competition focal point.SNP genotype and functional study thereof have become the integral part of post genome project.
Many methods about sudden change and single nucleotide polymorphism detection are arranged at present, as dna sequencing, restriction enzyme digestion length polymorphism, single strand conformation polymorphism, tetra-sodium order-checking and allele specific oligonucleotide oligonucleotide hybridization etc.Though these technology can be finished the detection to sudden change or nucleotide polymorphisms to a certain extent, at first need target fragment is increased to improve the sensitivity of detection, the site of their detections is quite limited like this, and therefore the information that obtains is incomplete.Develop method simple, cheap, sensitive, that analyze complete genome DNA information reliably for having great importance undoubtedly from full gene level understanding life.
Summary of the invention
Technical problem: the purpose of this invention is to provide the fixing means of a kind of full genome rolling circle amplification and product thereof, obtain when this method has realized full genomic information, saved the template preparation time greatly; Simultaneously, both kept the unit molecule number after the segmentization behind fragmentation, and improved again and used the sensitivity that detects because complete genomic information is amplified with the amplification of unit molecule form simultaneously.
Technical scheme: the present invention earlier with full genome with fragmentation, and the nucleic acid of fragmentation amplified with monomolecular form amplification, constitute the complete genome DNA chip, the full genomic information that makes acquisition accurately, reliable.
The fixing means of full genome rolling circle amplification of the present invention and product thereof is: genomic dna becomes short fragment through enzymic digestion or ultrasonication, the DNA of fragmentation connects connexon under the effect of enzyme, and connect into ring, with this toroidal molecule is that template is carried out rolling circle amplification, obtains the genomic fragment nucleotide sequence of rolling circle amplification; The genomic fragment nucleotide sequence of rolling circle amplification of dilution is fixed on a mechanically resistant material carrier surface by chemical process, make each genomic fragment nucleotide sequence occupy an independently position coordinates and separate fixing with other sequence at this carrier surface, thereby realize the fixing of whole genome sequence, constitute the complete genome DNA chip.After the genomic fragment nucleotide sequence is genomic fragmentization, connection, cyclisation, obtain having the genomic fragment nucleotide sequence that some toroidal molecules repeat pulsating unit molecule rolling circle amplification with universal primer rolling circle amplification toroidal molecule.The genomic fragment nucleotide sequence fixedly be chemical reaction between the genomic fragment nucleotide sequence of group alive by carrier surface and modification group, or the gel method that adopts the genomic fragment nucleotide sequence modified to aggregate into is fixed.Connexon be meant with genome in any segment all inequality, do not constitute yet with genome in one section oligonucleotide sequence of any segment complementary, an end or the two ends of fragmentation nucleic acid molecule all can connect connexon.The concentration of the genomic fragment nucleotide sequence of the rolling circle amplification of dilution guarantees do not have two nucleic acid molecule to occupy same position coordinates.The mechanically resistant material carrier surface contains amino or vitamin H active group, and the active group of connexon can be aldehyde radical or avidin or acrylamide group; Wherein, have at least an end of a connexon to have an active group, in the fixation procedure of fragmentation nucleotide sequence, can with carrier surface generation chemical reaction.
The complete genome DNA micro-array chip that makes up can be used for foranalysis of nucleic acids such as fluorescent hybridization, order-checking.This technology is assigned to genomic full detail in each dna fragmentation, and utilizes PCR method that the genome all information has been realized amplification, makes complete genomic analysis become simple.
Beneficial effect: the present invention compared with prior art has following advantage:
1, great advantage of the present invention is to obtain when having realized full genomic information, has saved the template preparation time greatly; Simultaneously, both kept the unit molecule number after the segmentization behind fragmentation, and improved again and used the sensitivity that detects because complete genomic information is amplified with the amplification of unit molecule form simultaneously,
2, the present invention adopts general connexon to realize being connected of fragmentation nucleic acid and amplification with general amplimer, can save a large amount of detection costs.
3, the purpose of this invention is to provide a kind of complete genome DNA microarray, obtain individual full genomic information by analysis to the complete genome DNA microarray, simultaneously (for example order-checking, hybridization analysis etc.) are realized the obtaining of information in several ways, thereby provide fabulous technical Analysis platform for the high throughput testing of genomic information entirely.
Description of drawings
Fig. 1 is the synoptic diagram of full gene rolling circle amplification of the present invention and product product fixing means thereof.
Fig. 2 is the structural representation of the genomic fragment behind the connection connexon among the present invention.
Have among the above figure: the ligation B of restriction enzyme digestion reaction A, ligase enzyme I, the ligation C of ligase enzyme 11, rolling circle amplification reaction D; Roll the fixation reaction E of ring product
Genomic dna 1, genomic fragment nucleotide sequence 2, connexon 3, toroidal molecule 4, rolling circle amplification reaction primer 5, amplifying nucleic acid sequence repeat sheet 6, plane surface 7.
Embodiment
The invention will be further described below with reference to accompanying drawing.
Genomic dna among the present invention forms short fragment through suitable digestion with restriction enzyme (perhaps ultrasonication), the DNA of fragmentation is connected with general connexon under the effect of enzyme, the nucleotide sequence of these fragmentations is connected into ring under the effect of ligase enzyme, use universal primer to carry out rolling circle amplification as template then with toroidal molecule, the DNA of fragmentation realizes the amplification amplification with monomolecular form, and the dilution amplified production makes the nucleic acid molecule of each fragmentation occupy an independently coordinate position.Thereby realized whole genome sequence fixing with the fragmentation form, constitute the complete genome DNA chip, this chip can be used for foranalysis of nucleic acids such as hybridization, order-checking, because the DNA of fragmentation is amplified with the amplification of unit molecule form, has therefore improved and has used the sensitivity that detects.
Full gene rolling circle amplification of the present invention and product product fixing means thereof take following scheme to realize:
With restriction enzyme (perhaps ultrasonication) genome is cut into size earlier and be the segment of 50-1000 base, and under the effect of ligase enzyme, these fragmentation nucleotide sequences are connected with a pair of general connexon, one of them connexon is complementary fully with amplification reverse primer sequence;
Obtain having some toroidal molecules with universal primer rolling circle amplification toroidal molecule and repeat pulsating unit molecule amplifying nucleic acid sequence sheet;
The unit molecule amplifying nucleic acid sequence sheet of dilution rolling circle amplification also is fixed at a mechanically resistant material carrier surface.Then whole genome sequence is with the form of the fragmentation plane surface that is fixed respectively, thereby has been built into the complete genome DNA micro-array chip.
Among Fig. 1, the genomic dna 1 that places the Eppendorff pipe is by using restriction enzyme digestion A, genomic fragment is turned to the genomic fragment nucleotide sequence 2 of big or small 50-1000 base, these fragmentation nucleotide sequences (B) under the effect of ligase enzyme I are connected 3 with a pair of general connexon, and one of them general connexon and rolling circle amplification reaction primer are complete complementation (referring to accompanying drawings 2).Fragmentation nucleotide sequence C under the effect of ligase enzyme that connecting arm connects connects into toroidal molecule 4, after adding rolling circle amplification reaction primer 5 and being the general connexon annealing of complete complementary, carry out rolling circle amplification reaction D, the unit molecule amplifying nucleic acid sequence sheet 6 of dilution rolling circle amplification also is fixed by chemical reaction at a mechanically resistant material carrier surface.Then whole genome sequence is with the form of the fragmentation plane surface 7 that is fixed respectively, thereby has been built into the complete genome DNA micro-array chip.
Each genomic fragment that comprises behind the connexon is made of two portions again among Fig. 2: genomic fragment nucleotide sequence 2, be used for rolling circle amplification reaction primer 5 be to be complementary to connexon 2 fully.
Example 1 yeast full genome fixing method
With reference to accompanying drawing 1,2, on a smooth sheet glass, hydrophobic treatment is carried out on the microwell plate surface with dichlorodimethylsilane.Micropore through washing, dry back with 5% 3-TSL 8330 acetone modify 1 hour, 5% glutaraldehyde processing after 2 hours carrier surface derive the aldehyde radical active group.
On the other hand, the yeast genes group is cut into the segment that size is about 500 bases with enzyme, and under the effect of ligase enzyme, these fragmentation nucleotide sequences are connected with a pair of general few nucleic acid molecule, the sequence of one of them general few nucleic acid molecule and rolling circle amplification primer is complementary fully.
The fragmentation nucleotide sequence that connecting arm connects connects into toroidal molecule under the effect of ligase enzyme, add amido modified rolling circle amplification reaction primer and carry out the rolling circle amplification reaction, the amido modified unit molecule amplifying nucleic acid sequence of dilution rolling circle amplification, make the mononucleotide molecule of each fragmentation occupy an independently coordinate position, the mononucleotide molecule of fragmentation is fixed on the slide and the carrier surface reaction with aldehyde radical active group, then the yeast whole genome sequence is with the form of the fragmentation plane surface that is fixed respectively, thereby has been built into yeast complete genome DNA micro-array chip.Utilize (for example order-checking, the hybridization analysis etc.) realization in several ways of this complete genome DNA chip to the analysis of the full genomic information of yeast.
Example 2 people's full genome fixing methods
With reference to accompanying drawing 1,2.
On the one hand, it is the segment of 50-1000 base that the people's gene group is become size with enzyme cutting (perhaps ultrasonication), and under the effect of ligase enzyme, these fragmentation nucleotide sequences are connected with a pair of general connexon, the sequence of one of them general few nucleic acid molecule and rolling circle amplification primer is complementary fully.
The fragmentation nucleotide sequence that connecting arm connects connects into toroidal molecule under the effect of ligase enzyme, behind the rolling circle amplification primer annealing that adding acrylamide group is modified, carry out the rolling circle amplification reaction.
Acrylamide modification rolling circle amplification reactant dilutes, is mixed into prepolymer solution with 3% acrylamide monomer, and be uniformly distributed on the clean sheet glass, make the mononucleotide molecule of each fragmentation occupy an independently coordinate position, be placed in the vacuum then, and under the vaporization atmosphere of Tetramethyl Ethylene Diamine, form gel with short its polymerization reaction take place, the mononucleotide molecule of fragmentation is fixed on the slide.Thereby be built into people's complete genome DNA micro-array chip.Utilize (for example order-checking, the hybridization analysis etc.) realization in several ways of this complete genome DNA chip to the analysis of the full genomic information of people.
Claims (6)
1, the fixing means of a kind of full genome rolling circle amplification and product thereof, it is characterized in that genomic dna becomes short fragment through enzymic digestion or ultrasonication, the DNA of fragmentation connects connexon under the effect of enzyme, and connect into ring, with this toroidal molecule is that template is carried out rolling circle amplification, obtains the genomic fragment nucleotide sequence of rolling circle amplification; The genomic fragment nucleotide sequence of rolling circle amplification of dilution is fixed on a mechanically resistant material carrier surface by chemical process, make each genomic fragment nucleotide sequence occupy an independently position coordinates and separate fixing with other sequence at this carrier surface, thereby realize the fixing of whole genome sequence, constitute the complete genome DNA chip.
2, the fixing means of full genome rolling circle amplification according to claim 1 and product thereof, after it is characterized in that the genomic fragment nucleotide sequence is genomic fragmentization, connection, cyclisation, obtain having the genomic fragment nucleotide sequence that some toroidal molecules repeat pulsating unit molecule rolling circle amplification with universal primer rolling circle amplification toroidal molecule.
3, the fixing means of full genome rolling circle amplification according to claim 1 and product thereof, what it is characterized in that the genomic fragment nucleotide sequence fixedly is chemical reaction between the genomic fragment nucleotide sequence of group alive by carrier surface and modification group, or the gel method that adopts the genomic fragment nucleotide sequence modified to aggregate into is fixed.
4, the fixing means of full genome rolling circle amplification according to claim 2 and product thereof, it is characterized in that connexon be meant with genome in any segment all inequality, do not constitute yet with genome in one section oligonucleotide sequence of any segment complementary, an end or the two ends of fragmentation nucleic acid molecule all can connect connexon.
5, the fixing means of full genome rolling circle amplification according to claim 1 and product thereof is characterized in that the concentration of the genomic fragment nucleotide sequence of the rolling circle amplification that dilutes guarantees do not have two nucleic acid molecule to occupy same position coordinates.
6, the fixing means of full genome rolling circle amplification according to claim 1 and product thereof, it is characterized in that the mechanically resistant material carrier surface contains amino or vitamin H active group, and the active group of connexon can be aldehyde radical or avidin or acrylamide group; Wherein, have at least an end of a connexon to have an active group, in the fixation procedure of fragmentation nucleotide sequence, can with carrier surface generation chemical reaction.
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| CNB2006100968427A CN100510106C (en) | 2006-10-20 | 2006-10-20 | Full genome rolling circle amplification and product fixing method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263830A (en) * | 2014-09-25 | 2015-01-07 | 徐州医学院 | Method for detecting nucleic acid molecules based on acrylamide gel chip |
| CN112739829A (en) * | 2018-09-27 | 2021-04-30 | 深圳华大生命科学研究院 | Construction method of sequencing library and obtained sequencing library and sequencing method |
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2006
- 2006-10-20 CN CNB2006100968427A patent/CN100510106C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263830A (en) * | 2014-09-25 | 2015-01-07 | 徐州医学院 | Method for detecting nucleic acid molecules based on acrylamide gel chip |
| CN112739829A (en) * | 2018-09-27 | 2021-04-30 | 深圳华大生命科学研究院 | Construction method of sequencing library and obtained sequencing library and sequencing method |
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| CN100510106C (en) | 2009-07-08 |
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