CN100432233C

CN100432233C - PCR method capable of eliminating non specific braid and RT-PCR chip prepared using said method

Info

Publication number: CN100432233C
Application number: CNB2004100450315A
Authority: CN
Inventors: 杨惠龄; 廖端芳; 徐阳炎; 罗迪贤; 李凯
Original assignee: University of South China
Current assignee: Nanhua University; University of South China
Priority date: 2004-07-09
Filing date: 2004-07-09
Publication date: 2008-11-12
Anticipated expiration: 2024-07-09
Also published as: CN1718737A

Abstract

The present invention discloses a PCR method for eliminating non-specific bands and precisely obtaining a target gene by a one-step method, and an RT-PCR chip prepared by the method. The method specifically comprises: when a PCR (RT-PCR or PCR) target gene is designed, one or more basic groups at the end of a primer 3' are acidified and decorated by thiophosphate; a polymerase used for extension reactions is a DNA polymerase with high Pfu fidelity. The DNA polymerase with high Pfu fidelity and strongest 3'-5 exonuclease activity can identify and close any one or more extension reactions with unmatched basic groups within the end 8 bp of the primer 3', and therefore, only templates which are completely matched with the end 8 bp of the primer 3' are extended in various PCR reactions. High fidelity with the templates is kept during extension. The present invention builds a technology platform which has the advantages of multiple purposes, high precision and high flux and is suitable for obtaining specific gene expression (including code area, transcription group and expression pattern comparison) and diagnosis by a chip technology.

Description

A kind of PCR method of eliminating non-specific band

Technical field

The present invention relates to a kind ofly be applicable to that chip technology obtains specific gene and expresses (comprise the coding region, transcribe group, express spectra relatively), PCR (RT-PCR, the PCR) method of diagnosis and the RT-PCR chip that utilizes this method to prepare, particularly a kind ofly eliminate the PCR method that non-specific band, single stage method accurately obtain goal gene, obtain the technology platform that specific gene is expressed (comprise the coding region, transcribe group, express spectra relatively), diagnosed to make up a high precision, high-throughput.

Background technology

Along with fulfiling ahead of schedule of human genome DNA's working draft sequence, the mankind enter the genome times afterwards comprehensively.In the genome times afterwards comprehensively, at first require to utilize human genome DNA's sequence information to study the structure and the function of gene and protein thereof, further then be that it takes place at cell and tissue, the functional mechanism under the development, old and feeble, dead and normal and morbid state.Every class cell or tissue is only expressed a cover special genes (but may change to some extent at irriate or differentiation phase), because generally many biological characteristicses depend on the synergy of range gene expression level, the research that changes at term single gene just demonstrates significant limitation.Have only differential expression, abundance difference, the dynamic change of comprehensive grasp range gene different times, could understand the interactive relationship of genotype and phenotype. thereby need a kind of extensive, high-throughout research strategy badly.Biochip technology is as a kind of emerging biometric technology, though the various countries scholar has seen its significant application value, because its high false positive rate, mispairing rate, expensive, low repeatability make this technology rapid development and widespread use be subjected to certain restriction.

Theoretically, rationally using length is the primer of 17 Nucleotide, and its expected frequence is on average every 4 ¹⁷=17 179 869 184bp just have a binding site (or one section homologous sequence), its length has surpassed more than 5 times of human DNA total length, so as seen the primer of 17 Nucleotide only has a binding site on human genome. use the primer of 17 Nucleotide that the human genome DNA is made pcr amplification, just might obtain single specific amplification band.Yet, true really not so, and since the high susceptibility of PCR reaction, the not strict coupling of primer and template, and extension also can carry out, and can not eliminate non-specific responding even improve annealing temperature again.Therefore, the conventional round pcr primer extension reaction by the Taq enzyme mediation that does not have 3 '-5 ' 5 prime excision enzyme activity non-specific band can occur in DNA cloning.The extension of PCR is to hold since 3 ' of two primers, and is determining the specificity of PCR product; 5 ' end is then limiting the length of PCR product.The Taq enzyme that does not have 3 '-5 ' 5 prime excision enzyme activity of Shi Yonging does not have correct functioning in the past, and the specificity of PCR product is determined by annealing temperature.Find in practical study: in the primer extension reaction of Taq enzyme or the mediation of other low-fidelity DNA polyase, 3 ' terminal pairing all can be extended in the annealing region of large span with unpaired primer, often needs to improve the specificity that annealing temperature improves product.Yet the annealing temperature that extension product fidelity requires in the differential responses system is different.In the annealing region of High variation, the mediation of low-fidelity DNA polyases such as Taq enzyme can't be removed, therefore can't be suitable for the requirement of homogeneous response condition in the chip technology, also be to cause the DNA product error rate that increases in the pilot chip or the false positive rate immediate cause up to 30%-50%.In recent years the Pfu high-fidelity DNA of Fa Xianing has strict template dependency and 3 '-5 ' 5 prime excision enzyme activity (proofreading activity), can proofread and correct enzymic activity with 3 '-5 ' according to template and a plurality of unpaired bases of primer 3 ' end are corrected into amplify non-purpose product with template matches, and in the annealing region of broad, still be suitable for.So list non-specific band can occur equally with the high-fidelity DNA polymerase-mediated primer extension reaction of Pfu, this also is that such high-fidelity DNA polymerase can not widely used defective.

No matter in vivo or external, polysaccharase is kept and duplicated fidelity is to proofread and correct enzyme active sites and the consistent result of polymerase activity site hight coordinate; Guiding chain combination template and polysaccharase in conjunction with after, polymerization or duplicate the tendency beginning.Wherein duplicate the height fidelity in the body and mainly be by proofreading and correct enzyme and cut the correct functioning of avtive spot and realize.By proofread and correct the enzyme point of contact examine the guiding chain whether with template matches, and will be not do not correct to such an extent that begin reproduction process after consistent with template with the guiding chain of template matches, this all has performance causing and duplicate in extension process.And to incorrigible mistake in the polymerization process, but closing of initiated polymerization or reproduction process then, thus it is consistent with the template height to keep filial generation.

Summary of the invention

The purpose of this invention is to provide and a kind ofly can accurately obtain the purpose product, effectively eliminate the PCR method of non-specific band.Utilize the present invention, can be accurately to genetic expression increase, detection and quantitative analysis, can be used for preparing high precision, high-throughput PCR (RT-PCR, PCR) chip, to be used to detect genetic expression.

In order to realize the foregoing invention purpose, the technical solution adopted in the present invention is: a kind of PCR method of eliminating non-specific band, have conventional PCR basic step, it is characterized in that when the design of primers of goal gene, primer 3 ' terminal one or more bases adopt the thiophosphoric acid modification; The used polysaccharase of extension is the Pfu high-fidelity DNA polymerase with 3 '-5 ' the strongest 5 prime excision enzyme activity.

Aforesaid method can be used for preparing the RT-PCR chip, to be used to detect genetic expression (comprising whether genetic expression reaches expression abundance detection by quantitative).Concrete grammar is that the primer downstream is totally 4 groups of the A/G/C/T of PolyA18-20+3 ' terminal bases thio-modification; The primer upstream stops the coding region design according to range gene, add fluorescence or other detectable marker in its every hole reaction and carry out that the primer extension reaction product is carried out in good time, detection by quantitative, with product have or not or whether how many indicators are expressed and are reached gene expression abundance.

Detection means of the present invention is used SYBR Green I fluorescence dye, with in good time PCR instrument of fluorescence or conventional agarose electrophoresis.

Because the Pfu high-fidelity DNA polymerase has strict template dependency and 3 '-5 ' 5 prime excision enzyme activity (correct functioning), can effectively discern primer 3 ' terminal one or more unpaired bases, add the characteristic that the anti-enzyme in primer 3 ' terminal sulfuration modification back is cut, therefore Pfu high-fidelity DNA polymerase 3 '-5 ' 5 prime excision enzyme activity can not be with the primer 3 ' Bottoming through the thiophosphoric acid modification, make the Pfu high-fidelity DNA polymerase to match or base mismatch is corrected to primer 3 ' end, and final " pass " closes the DNA polyreaction of primer and template mispairing, only allow primer 3 ' end 8bp polymerization complete and template matches to be extended, and in extension, keep height fidelity with template, finally obtain special purpose product.This method can be referred to as to realize " ON/OFF " mechanism of PCR (PT-PCR, PCR) product specificity efficient.Because the Pfu high-fidelity DNA polymerase with 3 '-5 ' the strongest 5 prime excision enzyme activity can be discerned and close in primer 3 ' the terminal 8bp any or the unmatched extension of a plurality of base.Therefore in various PCR reactions, this method only allow with primer 3 ' terminal 8bp in fully the template of coupling extended, and in extension, keep height fidelity with template.Use this method and can eliminate non-special product, the purpose product occurs with double chain form.And but SYBR Green I fluorescence dye specific combination dna double chain sends fluorescence, therefore available fluorescent PCR instrument to product carry out in good time, the accurate quantification analysis, thereby but high-throughput, collimation, the whole genetic transcription group of high accuracy analysis, expression group and tomb because of organizing (height of the having or not of genetic expression, abundance).

The present invention compares with existing PCR method, has following advantage and effect:

1, the present invention is to once important improvement traditional, that produce PCR (RT-PCR, the PCR) method of non-specific band, and its advantage is to eliminate non-specific band, high special.

2, the molecular biology research field be can be widely used in, amplification, detection, the quantitative analysis of genetic expression among the PCR (RT-PCR, PCR) are particularly useful for.

3, owing to broken through the limitation that the non-specific product of low-fidelity DNA polyase mediation such as in the annealing region of High variation Taq can't be removed, the present invention can be used for preparing high precision, high-throughput PCR (RT-PCR, PCR), can effectively remove error rate or false positive rate in the pilot chip amplification, be used to detect genetic expression, comprise coding region/transcribe group and express spectra relatively.

4, the present invention is simple, economic, practical, is convenient to promote.

Description of drawings

But Fig. 1 is the example that non-specific band, single stage method accurately obtain the purpose expressing gene among the specificity removal PCR (RT-PCR, PCR).

Embodiment

The invention will be further described below in conjunction with the drawings and specific embodiments.

Utilize the present invention that the Gaveolin-1 gene is increased.3 (M) expression Marker among Fig. 1,

band

1 and 2 is represented gene Gaveolin-1 total length and fragment respectively.

Concrete steps are as follows:

The first step: the extraction of mouse Gaveolin-1 (little recessed albumen 1) mRNA and cDNA's is synthetic, carries out according to ordinary method;

Second step: design of primers.Four kinds of primers of present embodiment design:

Primer I: the primer (band 14-15,18) that 3 ' end does not match and the end sulfuration is modified;

Primer I I:3 ' end does not match and vulcanizes the primer (band 4-9) of modification;

Primer I II:3 ' end mates and the primer (band 12-13) of end sulfuration modification;

The primer that primer I V:3 ' end coupling and sulfuration are modified (band 1-2,10-11,16-17).

The 3rd step: the selection of archaeal dna polymerase.

For relatively high-fidelity DNA polymerase and Lo-Fi polysaccharase Different Effects in RT-PCR to product, corresponding respectively selection Pfu high-fidelity DNA polymerase (band 1-2,4-5,8-9,14-17) with Taq Lo-Fi polysaccharase (band 6-7,10-13,18).

The 4th step: RT-PCR.

Reaction conditions is the reaction conditions of common RT-PCR: 94 ℃ of pre-sex change 3 minutes, and 94 ℃ of sex change 30 seconds, 52 ℃ or 60 ℃ of annealing 30 seconds, 72 ℃ were extended 45 seconds, and circulated 36 times.

The 5th step: agarose gel electrophoresis.

Carry out gel strength 2.0% according to a conventional method.

The result: show among Fig. 1 that the Pfu polysaccharase is in conjunction with primer 3 ' terminal thio-modification, the extension products of pairing primer presents single band (

band

1,2,16,17), and non-specific band is seen at the end; (band 4-5 8-9), presents high degree of specificity and any band does not appear in unpaired primer.In the extension of Taq enzyme mediation, no matter whether primer matches (band 10-13 and 6-7,18) or the primer all visible non-specific band of thio-modification (band 6-7,10-11 and 12-13,18) whether.

Claims

1, a kind of PCR method of eliminating non-specific band has conventional PCR basic step, it is characterized in that when the design of primers of goal gene, and primer 3 ' terminal one or more bases adopt the thiophosphoric acid modification; The used polysaccharase of extension is the Pfu high-fidelity DNA polymerase with 3 '-5 ' the strongest 5 prime excision enzyme activity.