CN101153338A - DNA sequencing method based on base modification protection reciprocation extension - Google Patents
DNA sequencing method based on base modification protection reciprocation extension Download PDFInfo
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- CN101153338A CN101153338A CNA2007101315507A CN200710131550A CN101153338A CN 101153338 A CN101153338 A CN 101153338A CN A2007101315507 A CNA2007101315507 A CN A2007101315507A CN 200710131550 A CN200710131550 A CN 200710131550A CN 101153338 A CN101153338 A CN 101153338A
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Abstract
A DNA sequencing method with protection on the reciprocated extension of the DNA based on base modification is provided, in which the sequencing of DNA is accomplished through the steps of 'extension of markers-cutting-protected extension' based on the base modification/protection. The method can realize the in situ elimination of the extension markers, while ensuring the completeness of the prolonged end of the primer extension, and gradually synthesizing/identifying the unknown bases, wherein, the sequencing method with one-by-one individual extension firstly extends a marked individual, secondly take the massage, and thirdly eliminate the individual and adds a protective individual, which is followed by derivation of the other individual, therefore, the no severe accumulation of the wrong extension, and the recognition of the sequence is accurate without the limits on the sequencing length.
Description
Technical field
The invention belongs to technical field of molecular biology; be particularly related to the former bit clear of a kind of realization and extend marker, protect the dna sequencing method that the primer extension end is complete, can progressively synthesize the unknown base of evaluation and relate to a kind of " mark extension-cutting-protection is extended " simultaneously.
Background technology
Prior art: in molecular biology research, the sequential analysis of DNA is the basis of further research and transformation goal gene.The sequencing technologies that is used for widespread use the most at present is the double deoxidating chain end cessation method of Sanger etc. (1977) invention, and the principle of Sanger method order-checking is exactly to utilize a kind of archaeal dna polymerase to extend the primer that is combined on the sequence template undetermined.Till mixing a kind of chain termination nucleotide.Sequencing is made of four independent reactions of a cover each time, and each reaction contains all four kinds of deoxynucleotide triphosphoric acids (dNTP), and sneaks into a kind of different dideoxyribonucleoside triphosphate (ddNTP) of limiting the quantity of.Extend needed 3-OH group because ddNTP lacks, the oligonucleotide of prolongation is optionally stopped at G, A, T or C place.Terminating point is decided by corresponding two deoxidations in the reaction.The relative concentration of each dNTPs and ddNTPs can be adjusted, and makes reaction obtain the chain termination product of group leader's hundreds of to several kilobase.They have the common starting point, but terminate on the different Nucleotide, can separate the fragment that varies in size by the high resolving power denaturing gel electrophoresis, and available x-ray film radioautograph or heterotope mark detect after the Gel Treatment.Nineteen ninety～1998 year, human genomic sequence finished and checking order amount to about 330Mb, account for about 11% of people's gene group; About 200 of the relevant genes of human diseases have been identified.In addition, the complete genomic order-checking of 17 kinds of biologies such as bacterium, archeobacteria, mycoplasma and yeast is finished.The advantage of Sanger method is to analyze unknown dna sequence dna, and unidirectional response to read Process capabi l i ty 32 longer, present technology can reach more than the 1000bp, then, in real work, a lot of situations need be carried out the sequence checking to the dna fragmentation of known array or analysis verification is carried out in site few in number, and in this case, the high-flux sequence method of shorter sequence is more suitable.
Sequencing technologies can be used in a plurality of fields, aspect molecular diagnostics, the Rapid identification that can be used for pathogenic micro-organism, the inherited disease molecular diagnosis is (as SNPs, bit frequencies such as SNP), aspect legal medical expert's evaluation, analyze as HVI and HVII hypervariable region plastosome D-Loop district, aspect pharmacogenomics, cooperate SNP to angiotensin-conerting enzyme (ACE) as Sweden Eurona Medical AB and Pyrosequencing company and analyze and many application are all being arranged aspect the agro-ecology.
At gene order-checking, developed several different methods at present, comprise improved afterwards capillary electrophoresis sequencing technologies from the gel electrophoresis determination techniques that begins at first based on the Sanger sequencing, DNA sequencing by hybridization (SBH to non-gel electrophoresis sequencing technologies, sequencing by hybridization) and flight time mass spectrum technology (time-of-flight mass spectrometry), arrive again earlier monster chip sequencing technologies and tetra-sodium sequencing technologies and some other single-molecule sequencing technology of Solexa company, in March, 2006, the president of Solexa company has just proposed the opinion that gene order-checking is risen again again, really not only there is continuous technology (and related article) to come out afterwards, and Luo Shi has also released GS20 tetra-sodium sequencing system under the strong backing of 454 Corp., when human genome order-checking in 2003 is finished, America NI H subordinate's state-run human genome research institute (NHGRI) has represented the whole nation is carried out the exploitation of revolutionary dna sequencing technology, and 1000 dollars of genome plans have been released thereupon, as seen sequencing technologies more and more receives publicity, indicating sequencing technologies invariably in the near future, it is not only a instrument as a kind of scientific research, and will be to an industry development, to better be human health service, more press close to human life.
Summary of the invention
Technical problem: the present invention is directed to above-mentioned technical problem, the dna sequencing method based on the base modification protection reciprocation extension is provided, this method can realize that former bit clear extends marker, protect simultaneously the primer extension end complete, can progressively synthesize the unknown base of evaluation.
Technical scheme: a kind of dna sequencing method based on the base modification protection reciprocation extension, determining of dna sequence dna by realizing based on base modification protection " mark extension-cutting-protection is extended " order-checking step, concrete order-checking step is: public fragment from one section 20-50 base length to dna profiling 3 to be determined ' end that introduce is as the sequencing primer hybridization sequences, will hybridize with the dna profiling to be determined on being fixed on solid phase carrier with the sequencing primer of 3 of public fragment complementation ' end base modification protection to combine; Mark extends: upwards go on foot in the sequenced dna template after gained is hybridized, order adds or adds simultaneously the labeled nucleotide monomer, on sequencing primer, carry out extension, and draw the base information of dna profiling to be determined in this time extended according to the labeled nucleotide monomer on extending; Cutting: the labeled nucleotide monomer that will go up on extending in the step is removed by cutting method; Protection is extended: through on extend identical with cut base modification protection nucleotide monomer on the sequencing primer sequence of step after handling; " mark extension-cutting-protection is extended " process that the circulation above-mentioned steps is carried out, the sequence of definite dna profiling to be determined thus.The modification protection nucleotide monomer is deoxyribonucleotide or the bi-deoxyribose Nucleotide that makes the modification protection of not cut group of monomer in the extension or particle.The modification protection nucleotide monomer is the deoxyribonucleotide or the bi-deoxyribose Nucleotide of sulfo-or iodo modification protection.The labeled nucleotide monomer is direct or indirect detected group or a particle on the mark on the nucleotide monomer.The labeled nucleotide monomer is that mark fluorescence molecule, radio isotope or horseradish peroxidase on the nucleotide monomer.The labeled nucleotide monomer is labeled dideoxynucleotide ribonucleotide or bi-deoxyribose Nucleotide.Cutting method is outer cutting method of enzyme or chemical chop method.The outer cutting method of the enzyme that utilizes exonuclease that cutting method is or utilize the chemical chop method of ammoniacal liquor, sodium hydroxide or hydrogen peroxide.The method that draws the base information of dna profiling to be determined in this time extended according to the labeled nucleotide monomer on extending is that fluoroscopic examination, chemiluminescence detection or radioautograph detect.
Beneficial effect:
The present invention compared with prior art has following advantage:
1. great advantage of the present invention is the method that has realized that marker is extended in the non-damaged in situ removing.In the synthetic sequence measurement of traditional extension, when marker has hindered the acquisition of further order-checking information, adopt usually with marker destruction or with the method for marker and monomer separation and remove marker information, further to extend order-checking.But, because marker all is a macromole usually, and marker all is stable covalent chemical bonds with monomeric the connection, therefore no matter be to adopt the method for chemistry or physics to destroy tag structure, still with marker and monomer cutting and separating, all will damage extending the site, its the possibility of result is that the marker macromole is because its space steric effect still hinders the carrying out of extension, also may be to cause the minimizing of extending the site, reduce next step extension signal, increase wrong information of extending, impact for order-checking length and exactness.And our method is removed be the marker monomer on extending; chemical bond rupture between monomer and the primer belongs to normal biological procedures; farthest protected and extended the integrity in site, and do not had the space steric effect influence further to extend order-checking.
2. the present invention is monomer extension order-checking one by one, extends a kind of labeled monomer, information extraction; remove this monomer then, add the modification protection monomer, a kind of labeled monomer down of deriving again; the storage effect that mistake is extended is not serious, and the interpretation of sequence is understood accurately, do not had the restriction of the length that checks order.
Description of drawings
Fig. 1 is the synoptic diagram that the present invention is based on the dna sequencing method of base modification protection reciprocation extension.Have among the figure: dna profiling 1., general thio-modification protection sequencing primer 2., solid phase carrier is 3.; a kind of labeled nucleotide monomer 4.; with 4. identical thio-modification protection nucleotide monomer 5., another kind of labeled nucleotide monomer 6., the chemical bond that can cut 7. and the chemical bond that can not cut 8..
Dna profiling and universal sequencing primer thing hybridization I; monomeric extension of a kind of labeled nucleotide and detection signal II; labeled nucleotide monomer II I on cutting is extended; modification protection nucleotide monomer IV in the extension; extend another kind of labeled nucleotide monomer and detection signal V; through III, the sequence information of dna profiling is determined in the circulation of IV and V the most at last.
Fig. 2 is the modification protection schematic diagram that the present invention is based on the dna sequencing method of base modification protection reciprocation extension.Have among the figure: thio-modification nucleotide monomer A, thio-modification 1., phosphorothioate dimer B, anti-cutting chemical bond 2..Thio-modification Nucleotide is meant that the Sauerstoffatom of normal oligodeoxynucleotide is replaced by sulphur atom, and then the chemically reactive of the chemical bond antienzyme of the thio-modification that forms, and then the monomer in the protection extension is not cut.
Embodiment
Preparation needs the dna profiling of order-checking; and be fixed; with general modification protection sequencing primer and dna profiling hybridization; by on sequencing primer, extending a kind of labeled nucleotide monomer, determine the sequence information of this position, the labeled monomer on will extending by the monomeric mode of cutting is then removed; extend subsequently and go up modification protection nucleotide monomer of the same race; guarantee the monomeric extension of a kind of labeled nucleotide down, circulation " mark extension-cutting-protection is extended " step is determined up to the dna profiling sequence.This method does not need to remove known signal by removing primer or destruction tag structure or fracture marker and monomeric chemical bond, and be to measure one by one, the storage effect that mistake is extended is not serious, and the interpretation of sequence is understood accurately, do not had the restriction of the length that checks order.
Fig. 1 is the synoptic diagram that the present invention is based on the dna sequencing method of base modification protection reciprocation extension, 1. dna profiling is fixed on solid phase carrier and 3. goes up, general modification protection sequencing primer 2. with dna profiling hybridization I, add a kind of labeled nucleotide monomer 4., under the polysaccharase effect, universal primer extended a kind of base II, and can determine by detecting whether dna profiling contains phase complementary base information with it in this extends; Remove the labeled monomer III that extends, remove known signal; Add thio-modification protection nucleotide monomer of the same race 5., in the downward thing IV that extends of polysaccharase effect, the site of further being checked order; Add another kind of labeled nucleotide monomer 6., on the basis of polysaccharase and modification protection nucleotide monomer, carry out next and plant determining of base; Circulation " mark extension-cutting-protection is extended " step is determined up to the dna profiling sequence.
Embodiment 1: measure people's gene group p16 exons 1 sequence based on the fluorescence sequencing of sulfo-base modification protection reciprocation extension
Design a pair of PCR primer at people's gene group p16 exons 1, wherein primer 5 ' terminal modified amino.With the p16 exons 1 sequence fragment in the PCR primer amplification people sample (blood, tissue etc.); behind the PCR product purification; way by point sample forms array on aldehyde group modified substrate; after amino and the aldehyde radical condensation, the unconjugated PCR product of flush away adopts sodium hydroxide (2%; w/v) way of sex change is removed loose DNA chain; obtain single template strand,, and guarantee that 3 ' distal process of template strand goes out 5～6 more than the base the primer and the hybridization of fixed dna profiling of modification protection.
Order according to dATP, dCTP, dGTP, dTTP, the each a kind of monomer of Cy5 mark and mixture (Cy5 labeled monomer concentration is 0.01 μ mol/L～10 μ mol/L) of unmarked monomeric certain proportion (100: 1～1: 100) of adding, with scanner write down this time extend in fluorescence have or not and strong and weak, determine to repeat the number of base by the linear relationship of setting up in advance.After collecting fluorescence data, cut away the fluorescence base dATP of extension with exonuclease III (1U/ μ L), add thio-modification dATP (0.01 μ mol/L～10 μ mol/L), under the effect of polysaccharase, fill the original position of Cy5-dATP, carry out the process that next step extends Cy5-dCTP then, so circulation is determined up to the PCR sequence.
Embodiment 2: measure people's gene group p16 exons 1 sequence based on the fluorescence sequencing of iodo base modification protection reciprocation extension
Design a pair of PCR primer at people's gene group p16 exons 1, wherein primer 5 ' terminal modified amino.With the p16 exons 1 sequence fragment in the PCR primer amplification people sample (blood, tissue etc.); behind the PCR product purification; way by point sample forms array on aldehyde group modified substrate; after amino and the aldehyde radical condensation, the unconjugated PCR product of flush away adopts sodium hydroxide (2%; w/v) way of sex change is removed loose DNA chain; obtain single template strand,, and guarantee that 3 ' distal process of template strand goes out 5～6 more than the base the primer and the hybridization of fixed dna profiling of modification protection.
Order according to dATP, dCTP, dGTP, dTTP, the each a kind of monomer of Cy5 mark and mixture (Cy5 labeled monomer concentration is 0.01 μ mol/L～10 μ mol/L) of unmarked monomeric certain proportion (100: 1～1: 100) of adding, with scanner write down this time extend in fluorescence have or not and strong and weak, determine to repeat the number of base by the linear relationship of setting up in advance.After collecting fluorescence data, cut away the fluorescence base dATP of extension with exonuclease III (1U/ μ L), add iodo and modify dATP (0.01 μ mol/L～10 μ mol/L), under the effect of polysaccharase, fill the original position of Cy5-dATP, carry out the process that next step extends Cy5-dCTP then, so circulation is determined up to the PCR sequence.
Embodiment 3: measure people's gene group p16 exons 1 sequence based on the radioautograph sequencing of sulfo-base modification protection reciprocation extension
Design a pair of PCR primer at people's gene group p16 exons 1, wherein primer 5 ' terminal modified amino.With the p16 exons 1 sequence fragment in the PCR primer amplification people sample (blood, tissue etc.); behind the PCR product purification; way by point sample forms array on aldehyde group modified substrate; after amino and the aldehyde radical condensation, the unconjugated PCR product of flush away adopts sodium hydroxide (2%; w/v) way of sex change is removed loose DNA chain; obtain single template strand,, and guarantee that 3 ' distal process of template strand goes out 5～6 more than the base the primer and the hybridization of fixed dna profiling of modification protection.
According to the order of dATP, dCTP, dGTP, dTTP, add a kind of phosphorus 32 (P at every turn
32) monomer of mark and the mixture (P of unmarked monomeric certain proportion (100: 1～1: 100)
32Labeled monomer concentration is 0.01 μ mol/L～10 μ mol/L), with autoradiographic technique write down this time extend in the isotropic substance signal have or not and strong and weak, determine to repeat the number of base by the linear relationship of setting up in advance.After collecting fluorescence data, cut away the P of extension with exonuclease III (1U/ μ L)
32DATP adds thio-modification dATP (0.01 μ mol/L～10 μ mol/L), under the effect of polysaccharase, fills the original position of Cy5-dATP, carries out next step then and extends P
32The process of-dCTP, so circulation is determined up to the PCR sequence.Embodiment 4: based on bi-deoxyribose Nucleotide the fluorescence sequencing of sulfo-base modification protection reciprocation extension measure people's gene group p16 exons 1 sequence
Design a pair of PCR primer at people's gene group p16 exons 1, wherein primer 5 ' terminal modified amino.With the p16 exons 1 sequence fragment in the PCR primer amplification people sample (blood, tissue etc.); behind the PCR product purification; way by point sample forms array on aldehyde group modified substrate; after amino and the aldehyde radical condensation, the unconjugated PCR product of flush away adopts sodium hydroxide (2%; w/v) way of sex change is removed loose DNA chain; obtain single template strand,, and guarantee that 3 ' distal process of template strand goes out 5～6 more than the base the primer and the hybridization of fixed dna profiling of modification protection.
Adding four kinds of different fluorescence modifications (is respectively Cy3, Cy5, FAM, the mixed solution of ddNTP TAMRA) (0.01 μ mol/L～10 μ mol/L) and polysaccharase (0.01 μ mol/L～10 μ mol/L), carry out the extension of a base at 3 of primer ' end, by scanning, the information of disposable definite first base of template sequence, cut away four kinds of different fluorescence base ddNTP of extension then with exonuclease III, order adds the mixture of four kinds of sulfo-bases (0.01 μ mol/L～10 μ mol/L) and polysaccharase (0.01 μ mol/L～10 μ mol/L), under the effect of polysaccharase, fill the original position of ddNTP, after cleaning up, add the ddNTP of four kinds of different fluorescence modifications and the mixed solution of polysaccharase again, carry out next step extension process, so circulation is determined up to the PCR sequence.
Claims (9)
1. dna sequencing method based on the base modification protection reciprocation extension is characterized in that determining by realizing that based on base modification protection " mark extension-cutting-protection is extended " order-checking step the step that specifically checks order is of dna sequence dna:
A. public fragment from one section 20-50 base length to dna profiling 3 to be determined ' end that introduce is as the sequencing primer hybridization sequences, will hybridize with the dna profiling to be determined on being fixed on solid phase carrier with the sequencing primer of 3 of public fragment complementation ' end base modification protection to combine;
B. mark extends: in the sequenced dna template after the hybridization of step a) gained, order adds or adds simultaneously the labeled nucleotide monomer, on sequencing primer, carry out extension, and draw the base information of dna profiling to be determined in this time extended according to the labeled nucleotide monomer on extending;
C. cutting: the labeled nucleotide monomer on extending in the step b) is removed by cutting method;
D. protection is extended: extend identical with cut base in step c) modification protection nucleotide monomer on the sequencing primer sequence after step c) is handled;
E. the above-mentioned steps that circulates b), c) and " mark extension-cutting-protection extend " process of d) being carried out, determine sequence of dna profiling to be determined thus.
2. a kind of dna sequencing method based on the base modification protection reciprocation extension according to claim 1 is characterized in that described modification protection nucleotide monomer is deoxyribonucleotide or the bi-deoxyribose Nucleotide that makes the modification protection of not cut group of monomer in the extension or particle.
3. a kind of dna sequencing method based on the base modification protection reciprocation extension according to claim 2 is characterized in that described modification protection nucleotide monomer is the deoxyribonucleotide or the bi-deoxyribose Nucleotide of sulfo-or iodo modification protection.
4. a kind of dna sequencing method based on the base modification protection reciprocation extension according to claim 1 is characterized in that described labeled nucleotide monomer is direct or indirect detected group or a particle on the mark on the nucleotide monomer.
5. a kind of dna sequencing method based on the base modification protection reciprocation extension according to claim 2 is characterized in that described labeled nucleotide monomer is that mark fluorescence molecule, radio isotope or horseradish peroxidase on the nucleotide monomer.
6. a kind of dna sequencing method based on the base modification protection reciprocation extension according to claim 1 is characterized in that described labeled nucleotide monomer is labeled dideoxynucleotide ribonucleotide or bi-deoxyribose Nucleotide.
7. a kind of dna sequencing method based on the base modification protection reciprocation extension according to claim 1 is characterized in that described cutting method is outer cutting method of enzyme or chemical chop method.
8. a kind of dna sequencing method based on the base modification protection reciprocation extension according to claim 7 is characterized in that outer cutting method of the enzyme that utilizes exonuclease that described cutting method is or the chemical chop method of utilizing ammoniacal liquor, sodium hydroxide or hydrogen peroxide.
9. a kind of dna sequencing method based on the base modification protection reciprocation extension according to claim 1 is characterized in that the described method that draws the base information of dna profiling to be determined in this time extended according to the labeled nucleotide monomer on extending is that fluoroscopic examination, chemiluminescence detection or radioautograph detect.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602719A (en) * | 2013-04-07 | 2014-02-26 | 北京迈基诺基因科技有限责任公司 | Gene sequencing method |
| CN103866010A (en) * | 2014-02-28 | 2014-06-18 | 郭诚 | Gene sequencing method |
| CN104611423A (en) * | 2009-11-16 | 2015-05-13 | 基因特力株式会社 | Genotyping method |
| WO2020199127A1 (en) * | 2019-04-02 | 2020-10-08 | 中国热带农业科学院热带生物技术研究所 | Design of sequencing primers and pcr-based method for sequencing whole genome |
| CN112575071A (en) * | 2020-12-25 | 2021-03-30 | 北京思尔成生物技术有限公司 | Method for directly performing Sanger sequencing on PCR amplification product without depending on purification |
-
2007
- 2007-09-14 CN CNB2007101315507A patent/CN100540682C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611423A (en) * | 2009-11-16 | 2015-05-13 | 基因特力株式会社 | Genotyping method |
| CN104611423B (en) * | 2009-11-16 | 2018-03-20 | 基因特力株式会社 | The method of Genotyping |
| CN103602719A (en) * | 2013-04-07 | 2014-02-26 | 北京迈基诺基因科技有限责任公司 | Gene sequencing method |
| CN103602719B (en) * | 2013-04-07 | 2016-06-08 | 北京迈基诺基因科技有限责任公司 | A kind of gene order surveying method |
| CN103866010A (en) * | 2014-02-28 | 2014-06-18 | 郭诚 | Gene sequencing method |
| CN103866010B (en) * | 2014-02-28 | 2016-04-20 | 郭诚 | A kind of method of gene sequencing |
| WO2020199127A1 (en) * | 2019-04-02 | 2020-10-08 | 中国热带农业科学院热带生物技术研究所 | Design of sequencing primers and pcr-based method for sequencing whole genome |
| CN112575071A (en) * | 2020-12-25 | 2021-03-30 | 北京思尔成生物技术有限公司 | Method for directly performing Sanger sequencing on PCR amplification product without depending on purification |
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| CN100540682C (en) | 2009-09-16 |
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