CN100429319C - Single tube nested type allele specific PCR method - Google Patents
Single tube nested type allele specific PCR method Download PDFInfo
- Publication number
- CN100429319C CN100429319C CNB2004100162816A CN200410016281A CN100429319C CN 100429319 C CN100429319 C CN 100429319C CN B2004100162816 A CNB2004100162816 A CN B2004100162816A CN 200410016281 A CN200410016281 A CN 200410016281A CN 100429319 C CN100429319 C CN 100429319C
- Authority
- CN
- China
- Prior art keywords
- primer
- minded
- equipotential
- polymerase chain
- base
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a single tube nested type polymerase chain reaction method for detecting mutation of a basic group by a super-short primer or a primer which contains a plurality of mismatched basic groups, specifically the present invention utilizes a super-short primer of a basic group whose length is from 6 to 14 or a primer which has the normal length and contains a plurality of mismatched basic groups to carry out allele specific amplification, which leads a wild allele specific primer to completely lose the capability of combining a saltant mould plate, and leads a saltant allele specific primer to completely lose the capability of combining a wild mould plate. Therefore, the false positive of the detection is overcome, and the reliability of the detection is improved. The single tube nested type polymerase chain reaction method is suitable for detection of nucleic acid of single basic group mutation by a molecular biology laboratory and medical clinic detection, and is especially suitable for nucleic acid analysis of hereditary diseases, variation virus infection, drug resistance bacterial infection, and metabolic diseases caused by single basic group mutation.
Description
Technical field
The present invention relates to Protocols in Molecular Biology, particularly relate to a kind of method that detects the polymerase chain reaction of the single-minded variation of equipotential.
Technical background
(single nucleotide polymorphism SNP), is meant that on genomic level because the caused dna sequence polymorphism of single nucleotide diversity, it accounts for human more than 90% of heritable various polymorphisms to single nucleotide polymorphism.Just there is a SNP in average per 500～1000 base pairs in the human genome according to estimates, the sum of SNP is up to millions of in 3,200,000,000 base pairs altogether, with the origin cause of formation of disease and the reaction of medicine etc. direct or indirect relation is arranged, SNP will will produce immeasurable influence to the research of population genetics, pharmacy industry, medical jurisprudence, cancer and heredopathia even evolution.
The technology a lot (Kwok, P.Y.ed, Single NucleotidePolymorphisms method and protocol, Humana Press, 2003) of detection single nucleotide polymorphism can be divided into several big classes but every class methods all exist various shortcomings and limitation.The medium position of each class methods single-minded PCR (AC-PCR) method is the simplest sensitive again, and still the wild primer of existing AC-PCR method and the single-minded sudden change template of equipotential often also can be annealed and be caused false positive, and vice versa.This makes existing various AC-PCR method confidence levels lower.Another kind of method is with various physics, chemistry and molecular biology method amplified production to be analyzed after taking turns pcr amplification one, comprise that traditional method such as dna sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP) are analyzed and the method for newly-developed such as mass spectroscopy, little sequencing, chemical cracking method, sex change high performance liquid chromatography (HPLC) etc.These methods exist amplified production to pollute, speed is slow, program is complicated, difficult automatization, cost is high and need problem such as Special Equipment all not to be widely adopted.The 3rd class methods relate to oligonucleotide hybridization, comprise method, dna microarray method and Invader Assay method etc. based on ligase enzyme, and these methods still are in the laboratory stage of fumbling and are not extensively promoted the use of by clinical.The real time fluorescence quantifying PCR method that emerges in the nineties is realizing having stepped major step aspect stopped pipe detection and the quantification, also has to be applied to the report that SNP detects.But use real-time method to detect SNP, exist and the same false positive problem of the single-minded PCR method of existing equipotential based on fluorescence green I.Using real-time method based on molecular beacon (hair clip formula probe) and 5 ' nuclease (TaoMan) to detect SNP then needs to design probe with various fluorophors, must be equipped with expensive real time fluorescent quantitative instrument simultaneously.
Summary of the invention
Technical problem to be solved by this invention
The present invention just provides the single-minded polymerase chain reaction method of a kind of single tube nested type equipotential, above-mentioned various detection SNP method amplified production pollutes, speed is slow, program is complicated to overcome, difficult automatization, cost is high and need shortcoming and limitation such as Special Equipment, develop a kind of not only simple, sensitive, quick and economy but also method accurately.
Inventive concept
The used a pair of primer length of the single-minded PCR method of existing equipotential is the 18-30 base, and except that mating fully with template at primer order the single-minded mutating alkali yl of equipotential.Though in this case the bonding strength of primer and matching template very high energy finish amplification smoothly, the bonding strength of the single-minded sudden change template of primer and equipotential is still quite high, with template annealing can take place and finish amplification and cause false positive under non-rigorous condition.The present invention is directed to this reason, adopt the single-minded primer of ultrashort equipotential or on the single-minded primer of equipotential, import some mispairing, wild primer and combining of matching template are still reached finish the needed intensity of amplification, the bonding strength of the single-minded sudden change template of wild primer and equipotential then is lower than the required threshold value of generation annealing.In order to overcome ultrashort primer and the non-single-minded amplification problem that contains the base mismatch primer, the invention provides from complicated genome such as human gene group DNA's enrichment To Template, and can finish the method for whole amplification procedure with single reaction tubes.When the single-minded sudden change of equipotential betides on simple template such as plasmid or the phage genome, the single-minded primer of ultrashort equipotential or contain the single-minded primer method of base mismatch equipotential and can directly use.Appear at as SNP on the exon of gene, the single-minded sudden change of equipotential will be copied on corresponding RNA and the cDNA, and it is the method for template detection SNP that the present invention is equally applicable to cDNA.The real-time fluorescence detection single nucleotide polymorphism method that the present invention program also is suitable for based on the green I of SYBR reduces its false positive.
Below with some concrete data principle of the present invention is described.Use professional primer-design software Oligo6.0 (by Molecular Biology Insights, Inc. produces).In people actomyosin gene, select 7 the 3 ' oligonucleotide that end is identical at random, calculate to its melting temperature(Tm) with the template bonding strength, it is big more to the influence of primer melting temperature(Tm) at 3 ' end base mispairing that the result shows that primer is got over short primer, make the relative reduction also big more (table 1 and table 2) of primer and the bonding strength of template, it is just strong more to illustrate that primer is distinguished the ability of matching template and the single-minded sudden change template of equipotential.
Base mispairing of table 1. is to the influence of primer melting temperature(Tm)
The statistics of table 2.18-30 base long primer and template bonding strength
The primer length scope of Standard PC R and the existing single-minded PCR of equipotential is the 18-30 base, wild (sudden change) primer and wild template (the single-minded sudden change template of equipotential) bonding strength average value ranges is 452-598, is significantly higher than needed bonding strength 360 points of very high preciseness primer (table 2 and 3).But, the single-minded sudden change template of wild (sudden change) primer and equipotential (wild template) bonding strength average value ranges is 220-308, the maximum that has all surpassed very low preciseness primer allows false bonding strength, wherein some reaches the desired bonding strength of very high preciseness primer, and so high bonding strength will cause false positive.
The primer preciseness standard that the professional primer-design software Oilgo6.0 of table 3 sets
| Preciseness | Very high | High | Medium | Still can | Low | Very low |
| With the template bonding strength | 360 | 340 | 320 | 310 | 300 | 290 |
| The false bonding strength of maximum permission | 160 | 170 | 180 | 190 | 200 | 200 |
If the primer of the single-minded PCR of equipotential is reduced to the 6-15 base, then wild (sudden change) primer and wild template (the single-minded sudden change template of equipotential) bonding strength average value ranges is 174-408 (table 4), most standards that reach medium preciseness primer in the oligonucleotide of being tested, wherein some has reached the needed bonding strength of very high preciseness primer and can finish annealing.And the single-minded sudden change template of wild (sudden change) primer and equipotential (wild template) bonding strength average value ranges is reduced to 64-184, most has been lower than 160 of false bonding strengths that very high preciseness primer allowed can not anneal (seeing Table 2 and 4)
The statistics of table 4.6-15 base long primer and template bonding strength
In primer, import some mispairing and can regulate the needs that the bonding strength of primer and coupling and the single-minded template of equipotential makes it coincidence detection SNP equally.Table 5 is used the Oligo6.0 result calculated for to select one 15 base oligonucleotide ATATCGCCGCGCTCG (5 ' to 3 ') at random in people actomyosin gene.The bonding strength of this primer and matching template is 505 points, and far above 360 required points of very high preciseness primer, but G takes place 3 ' base and C conversion back still has 216 points with the template bonding strength.If with the 7th G of primer change into C then primer and matching template bonding strength be 316 points, reach medium preciseness primer requirement, and the bonding strength of the single-minded sudden change template of primer and equipotential is reduced to 123 points, the Interference Detection of can not annealing again.Perpendicular expression of in the table 5 second is at the base of the single-minded sudden change of the equipotential bonding strength of wild primer and the single-minded sudden change template of equipotential during different positions in wild primer.Other data are the bonding strength of wild primer after other positions of wild primer import a base mismatch again and the single-minded sudden change template of equipotential.Two of different positions importings or more base mismatch at primer when primer length is the 18-30 base can reach same purpose.
Table 5. base mismatch is to the influence of primer and template bonding strength
Technical scheme
The invention provides the single-minded polymerase chain reaction method of a kind of single tube nested type equipotential, its steps in sequence comprises:
(1) with overcoat primer amplification 10-30 circulation;
(2) with interior cover primer amplification 10-30 circulation;
Wherein, the overcoat primer is and the rigorous paired primer of template that one of interior cover primer is that length is the ultrashort primer of 6-14 base or the mispairing primer that contains 1-30 base mismatch.
The length that the above-mentioned a kind of preferred version that utilizes the single-minded polymerase chain reaction method of equipotential that ultrashort primer carries out is ultrashort primer is the 8-12 base.
The single-minded sudden change of equipotential that the above-mentioned another kind of preferred version that utilizes the single-minded polymerase chain reaction method of equipotential that ultrashort primer advances is ultrashort primer designs in the 1-6 position of primer 3 ' terminal number, in primer, introduce 0-6 base mismatch simultaneously, be preferably introduced into 0-2 base mismatch.
The above-mentioned a kind of preferred version of the single-minded polymerase chain reaction method of equipotential that the mispairing primer carries out that utilizes is the long 15-50 base of mispairing primer, a preferred long 15-30 base, the single-minded sudden change of equipotential designs in the 1-6 position of primer 3 ' terminal number, in primer, introduce 1-30 base mismatch simultaneously, be preferably introduced into 2-10 base mismatch, base mismatch can be concentrated or disperse, and preferred every interval 5-8 base introduced a base mismatch.
Above-mentioned to utilize a kind of preferred version ultrashort or the single-minded polymerase chain reaction method of equipotential that the mispairing primer carries out be ultrashort or the bonding strength of mispairing primer and matching template greater than 250 points, be preferably greater than 300 points, and with the bonding strength of the single-minded sudden change template of equipotential less than 160 points, preferably less than 120 points.
The another kind of preferred version of the above-mentioned single-minded polymerase chain reaction method of equipotential be the melting temperature(Tm) of amplified production of overcoat primer than the high 2-10 of melting temperature(Tm) ℃ of the amplified production of interior cover primer, and the PCR reactions steps is according to this:
(1) with the above annealing temperature amplification 10-30 circulation of the highest permission annealing temperature of cover primer in being higher than;
(2) the control annealing temperature is below the melting temperature(Tm) of interior cover primer, and the control denaturation temperature is the melting temperature(Tm) of cover amplified production in the denaturation temperature that is lower than the overcoat amplified production is higher than, and continues amplification 10-30 circulation.
And, the fs overcoat and and subordinate phase in 2-3 round-robin transitory stage arranged between the cover reaction, this stage denaturation temperature is identical with the fs, and annealing temperature is identical with subordinate phase.
The another kind of preferred version of the above-mentioned single-minded polymerase chain reaction method of equipotential for the triple nested type primers of design and with ultrashort primer as the primer of interior cover.
The present invention finishes the detection of SNP with the single tube nested PCR, and the overcoat primer is that high preciseness primer is in order to enrichment target gene template.One of interior cover primer that is used for the single-minded amplification of equipotential is ultrashort primer or contains the base mismatch primer.For overcoming the non-single-minded amplification problem of ultrashort primer and the single-minded PCR of mispairing primer equipotential, at a pair of overcoat primer of the periphery design of the single-minded pcr amplification product of equipotential, overcoat primer extension product denaturation temperature likens to the single-minded amplified production melting temperature(Tm) of the equipotential of interior cover is high more than 2 ℃, and is preferred high more than 3 ℃; Control overcoat PCR reaction denaturation temperature is higher more than 2 ℃ than overcoat amplified production melting temperature(Tm), and cover PCR reaction denaturation temperature is between overcoat amplified production and interior cover amplified production in the control.The highest permission annealing temperature of cover primer is lower more than 2 ℃ than the working control annealing temperature of overcoat reaction in the design simultaneously, and is preferably low more than 3 ℃.Cover primer maximum permissible temperature in control overcoat reaction annealing temperature is higher than; Cover reaction in cover reaction annealing temperature is fit in the control.The fs overcoat and and subordinate phase in 2-3 round-robin transitory stage arranged between the cover reaction, this stage denaturation temperature is identical with the fs, and annealing temperature is identical with subordinate phase.
Beneficial effect
1. detect the single nucleotide polymorphism method with the single-minded PCR of existing various non-equipotential and compare, the inventive method is simple, sensitive, economical, quick.Neither need be at the stencil design fluorescent probe, there is not process behind the complicated time-consuming PCR yet, more do not need expensive detection equipment.
2. compare with the existing single-minded PCR method of various equipotentials, false positive of the present invention is low, and is with a high credibility.
3. the single-minded base mismatch of equipotential can be positioned on 3 ' end base of primer, also can be positioned on other bases of nearly 3 ' end, makes primer to design and selection bigger space be arranged.
4. in the single-minded primer of equipotential, introduce mispairing, not only reduce to detect false positive, also have more multimachine can choose the low and low primer of primer 3 ' end base complementrity of hairpin structure, help overcoming the formation of primer dipolymer.
Embodiment
Embodiment 1
Embodiment 1 is a target with people beta actomyosin gene (American National bioinformation center gene pool numbering BC016045), suppose that the single-minded sudden change of equipotential occurs in the 1281st base (changing into C by G) or the 1356th bit base (changing into G by C), primer order and characteristic are shown in table 6.A1 is an overcoat enrichment template primer, and A2 and A3 are the single-minded PCR primer of interior cover equipotential.The single-minded primer of the equipotential of A2 is positive primer, primer 3 ' terminal number rise the 3rd base at equipotential single-minded sudden change represent with black face; The single-minded primer of the equipotential of A3 is an anti-primer, primer 3 ' end base at equipotential single-minded sudden change represent with black face.
Table 6. embodiment 1 primer order and characteristic
| Title | The position | Length | Melting temperature(Tm) (℃) | With the template bonding strength | (5 ' to 3 ') in proper order |
| A1 just | 1132- 1159 | 28 | 81.3 | 462 | CAG CAG ATG TGG ATC AGC AAG CAG GAG T |
| A1 is anti- | 1399- 1426 | 28 | 79.5 | 532 | AAT CAA AGT CCT CGG CCACAT TGT GAA C |
| A2 is just wild | 1275- 1283 | 9 | 39.3 | 253 | TGG CAT GGC |
| A2 is just prominent | 1275- 1283 | 9 | 11.9 ＊ | 136 | TGG CAT CGC |
| A2 is anti- | 1380- 1389 | 9 | 36.4 | 246 | GCT CGC TCC |
| A3 just | 1187- 1196 | 10 | 38.9 | 315 | ACC GCA AAT G |
| A3 is anti-wild | 1356- 1365 | 10 | 36.1 | 282 | ACC TTC ACC G |
| A3 is anti-prominent | 1356- 1365 | 10 | 9.2 ＊ | 99 | ACC TTC ACC C |
*The melting temperature(Tm) that the most contiguous base method is calculated
Do not contain intron between the positive anti-primer of overcoat primer " A1 ", long 295 bases of amplified production, 84 ℃ of amplified production melting temperature(Tm)s; Long 114 bases of interior cover primer " A2 " amplified production, 78.4 ℃ of amplified production melting temperature(Tm)s; Long 179 bases of interior cover primer " A3 " amplified production, 79.7 ℃ of amplified production melting temperature(Tm)s.
The Advantage-2 test kit of Clontech company is adopted in embodiment 1 test, every tube reaction volume 5 microlitres, and positive each 0.4 μ M of anti-primer, per 100 microlitre reaction solutions add 0.5 microgram human gene group DNA.The PCR program is 94 ℃ of first sex change 1 minute, 94 ℃ of 10 second of preceding 3 circulation sex change, anneal and extend 72 ℃ 1 minute; 88 ℃ of 10 second of middle 17 circulation sex change, anneal and extend 72 ℃ 1 minute; Repeatedly 88 ℃ of 10 second of circulation sex change, anneal 31-55 ℃ of 30 seconds, extend 68 ℃ of 30 second; 82 ℃ of 10 second of last 14 circulation sex change, anneal 31-55 ℃ of 30 seconds, extend 68 ℃ of 30 second.Test-results is shown in table 7.
Table 7. embodiment 1 test-results
The result show primer to the positive open country/A2 of A2 anti-and A3 just/A3 is anti-wildly all to obtain the target amplification product in 31 to 50 ℃ of scopes, and A2 just anti-the and A3 of prominent/A2 just/A3 instead dashes forward primer to there is the formation of target amplification product under similarity condition.
Embodiment 2.
Embodiment 2 is a target with people beta actomyosin gene (American National bioinformation center gene pool numbering BC016045) also, supposes that the single-minded sudden change of equipotential occurs in the 1281st base (changing into C by G).Overcoat enrichment template is identical with embodiment 1 with primer.A4 is the single-minded PCR primer of interior cover equipotential, long 20 bases of primer, the single-minded primer of equipotential is positive primer, primer 3 ' terminal number rise second base at equipotential single-minded sudden change represent that with black face the 8th (the changing A into by T) and 16 (changing C into by G) of rising at 3 ' terminal number of the single-minded primer of equipotential introduced two base mismatch and represented with underscore.Primer order and characteristic see Table 8.
Table 8. embodiment 2 primers order and characteristic
| Title | The position | Length | Melting temperature(Tm) (℃) | With the template bonding strength | (5 ' to 3 ') in proper order |
| A4 is just wild | 1263- 1282 | 20 | 48.0 ＊ | 306 | ACA A GA TGA GAT TGG CAT GG |
| A4 is just prominent | 1263- 1282 | 20 | 42.2 ＊ | 137 | ACA ACA TGA GAT AGG CAT CG |
| A4 is anti- | 1380- 1393 | 14 | 62.5 | 371 | GGG ATG CTC GCT CC |
*The melting temperature(Tm) that the most contiguous base method is calculated
Long 131 bases of interior cover amplified production, 79.2 ℃ of melting temperature(Tm)s.Increase by embodiment 1 same procedure.Primer instead all obtains the target amplification product to the positive open country/A4 of A4 in 31 to 50 ℃ of scopes as a result, and primer to A4 just prominent/A4 instead do not have the target amplification product to form under similarity condition.
Embodiment 3
Embodiment 3 is a target with people's glyceraldehyde-3-phosphate dehydrogenase gene (American National bioinformation center gene pool numbering XM_006959).Suppose that the single-minded sudden change of equipotential occurs in the 415th base (changing into C by G).Primer order and characteristic are shown in table 9.G1 is an overcoat enrichment template primer, and G2 is the single-minded PCR primer of interior cover equipotential, and the single-minded primer of equipotential is positive primer, primer 3 ' terminal number rise the 5th base at equipotential single-minded sudden change represent with black face.
Table 9. embodiment 3 primers order and characteristic
| Title | The position | Length | Melting temperature(Tm) (℃) | With the template bonding strength | (5 ' to 3 ') in proper order |
| G1 just | 343- 366 | 24 | 81.4 | 536 | GCT GGC GCT GAG TAC GTC GTG GAG |
| G1 is anti- | 932- 958 | 27 | 83.4 | 481 | AGG TGG AGG AGT GGG TGT CGC TGT TGA |
| G2 is just wild | 412-4 19 | 8 | 35.6 | 301 | CAG GGG GG |
| G2 is just prominent | 412-4 19 | 8 | 8.5 ＊ | 116 | CAG CGG GG |
| G2 is anti- | 550-5 58 | 9 | 46.0 | 306 | GGC CAG GGG |
*The melting temperature(Tm) that the most contiguous base method is calculated
Amplified production length 616 bases of overcoat primer " G1 ", 90.4 ℃ of amplified production melting temperature(Tm)s.Interior cover primer " G2 " amplified production length 147 bases, 87.1 ℃ of amplified production melting temperature(Tm)s.
The pcr template of embodiment 3 is cDNA, and synthetic from people's total tissue RNA by Oligo (dT) with the reverse transcription test kit of Clontech, per 50 microlitre PCR reaction solutions add 5 microlitre cDNA.PCR method is identical with embodiment 1, PCR 94 ℃ of the sex change 1 minute that circulate first, 94 ℃ of 10 second of preceding 20 circulation sex change.Back 89 ℃ of 10 second of 15 circulation sex change.Preceding 20 cycle annealings of PCR and extend 72 ℃ 1 minute, back 13 cycle annealing 31-55 ℃ 30 seconds, extend 68 ℃ of 30 second.94 ℃ of 10 second of middle two circulation sex change, anneal 31-55 ℃ of 30 seconds, extend 68 ℃ of 30 second.Test-results is shown in table 10.
Table 10. embodiment 3 test-results
The result shows that primer instead all obtains the target amplification product to the positive open country/G2 of G2 in 31 to 45 ℃ of scopes, and primer to G2 just prominent/G2 instead do not have the target amplification product to form under similarity condition.
Claims (9)
1. single-minded polymerase chain reaction method of single tube nested type equipotential, the overcoat primer of described reaction is and the rigorous paired primer of template that one of interior cover primer is that length is the ultrashort primer of 6-14 base or the mispairing primer that contains 1-30 base mismatch; The melting temperature(Tm) of the amplified production of overcoat primer is than the high 2-10 of melting temperature(Tm) ℃ of the amplified production of interior cover primer, and the PCR reactions steps is followed successively by:
(1) with the above annealing temperature amplification 10-30 circulation of the highest permission annealing temperature of cover primer in being higher than;
(2) the control annealing temperature is below the melting temperature(Tm) of interior cover primer, and the control denaturation temperature is the melting temperature(Tm) of the amplified production of cover primer in the denaturation temperature that is lower than the amplified production of overcoat primer is higher than, and continues amplification 10-30 circulation;
And, the fs overcoat and and subordinate phase in 2-3 round-robin transitory stage arranged between the cover reaction, this stage denaturation temperature is identical with the fs, and annealing temperature is identical with subordinate phase.
2. the single-minded polymerase chain reaction method of equipotential according to claim 1, the length that it is characterized in that ultrashort primer is the 8-12 base.
3. the single-minded polymerase chain reaction method of equipotential according to claim 1 is characterized in that the single-minded sudden change of equipotential of ultrashort primer designs in the 1-6 position of primer 3 ' terminal number, introduces 0-6 base mismatch simultaneously in primer.
4. the single-minded polymerase chain reaction method of equipotential according to claim 3 is characterized in that 0-2 base mismatch of ultrashort primer introduction.
5. the single-minded polymerase chain reaction method of equipotential according to claim 1, it is characterized in that the long 15-50 base of mispairing primer, the single-minded sudden change of equipotential designs in the 1-6 position of primer 3 ' terminal number, introduces 1-30 base mismatch simultaneously in primer, and base mismatch can be concentrated or disperse.
6. the single-minded polymerase chain reaction method of equipotential according to claim 5 is characterized in that the long 15-30 of a mispairing primer base, introduces 2-10 base mismatch in the primer, and every interval 5-8 base introduced a base mismatch.
7. according to the single-minded polymerase chain reaction method of each described equipotential among the claim 1-6. the bonding strength that it is characterized in that ultrashort or mispairing primer and matching template is greater than 250 points, and with the bonding strength of the single-minded sudden change template of equipotential less than 160 points.
8. the single-minded polymerase chain reaction method of equipotential according to claim 8, the bonding strength that it is characterized in that ultrashort or mispairing primer and matching template be greater than 300 points, and with the bonding strength of the single-minded sudden change template of equipotential less than 120 points.
9. polymerase chain reaction method according to claim 1, it is characterized in that designing triple nested type primers and with ultrashort primer as the primer of interior cover.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100162816A CN100429319C (en) | 2004-02-13 | 2004-02-13 | Single tube nested type allele specific PCR method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100162816A CN100429319C (en) | 2004-02-13 | 2004-02-13 | Single tube nested type allele specific PCR method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1654671A CN1654671A (en) | 2005-08-17 |
| CN100429319C true CN100429319C (en) | 2008-10-29 |
Family
ID=34892222
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100162816A Expired - Fee Related CN100429319C (en) | 2004-02-13 | 2004-02-13 | Single tube nested type allele specific PCR method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100429319C (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1381588A (en) * | 2002-05-17 | 2002-11-27 | 徐定邦 | Polymerase chain reaction method using ultra-long primer to improve gene amplification efficacy |
| CN1384209A (en) * | 2002-05-23 | 2002-12-11 | 江西农业大学 | Specific primer sequence of cattle Y chromosome and application thereof in cattle embryo sex identification |
-
2004
- 2004-02-13 CN CNB2004100162816A patent/CN100429319C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1381588A (en) * | 2002-05-17 | 2002-11-27 | 徐定邦 | Polymerase chain reaction method using ultra-long primer to improve gene amplification efficacy |
| CN1384209A (en) * | 2002-05-23 | 2002-12-11 | 江西农业大学 | Specific primer sequence of cattle Y chromosome and application thereof in cattle embryo sex identification |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1654671A (en) | 2005-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7318072B2 (en) | Methods for variant detection | |
| US11118216B2 (en) | Nucleic acid analysis by joining barcoded polynucleotide probes | |
| EP2759601A1 (en) | Gene copy number variation measurement method | |
| CN106119363A (en) | SNP combination, detection method and test kit for the detection of antituberculotics hepatic injury susceptible genotype | |
| WO2017044993A2 (en) | Nucleic acid analysis by joining barcoded polynucleotide probes | |
| WO2007083766A1 (en) | Method for detecting nucleic acid sequence using intramolecular probe | |
| den Dunnen et al. | MLPA and MAPH: sensitive detection of deletions and duplications | |
| CN112029851A (en) | Method and kit for detecting gene polymorphism of clopidogrel medication and application of kit | |
| US20160289752A1 (en) | Method for the Isothermic Amplification of Nucleic Acid and SNP Detection | |
| EP2013366B1 (en) | Sequencing of the L10 codon of the HIV gag gene | |
| CN110172500B (en) | Isothermal typing method for single nucleotide polymorphism | |
| CN100429319C (en) | Single tube nested type allele specific PCR method | |
| CN114277108B (en) | Primer probe combination, kit and method for SNP locus detection | |
| JPWO2007055255A1 (en) | Method for amplifying a plurality of nucleic acid sequences for identification | |
| CN110741094A (en) | method for correcting amplification bias in sequencing of amplicons | |
| CN111041079A (en) | Flight mass spectrum genotyping detection method | |
| CN102424833A (en) | Real-time PCR gene detection chip for multiple gene mutation sites and detection method thereof | |
| CN104152568B (en) | High-throughput STR sequence core repeat number detection method | |
| WO2013053852A1 (en) | Design of stem-loop probes and utilization in snp genotyping | |
| EP3455376B1 (en) | Method for producing a plurality of dna probes and method for analyzing genomic dna using the dna probes | |
| CN112266950A (en) | A probe-primer combination and detection kit thereof | |
| CN112779322A (en) | Gene mutation detection kit based on non-fluorescence labeled probe and high-resolution melting curve, detection method and application thereof | |
| CN110938681A (en) | Allele nucleic acid enrichment and detection method | |
| CN114929893A (en) | Method for differential quantification of nucleic acids | |
| CN104152542B (en) | Tumor ABL1 gene loci parting detecting reagent, using method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20081029 Termination date: 20110213 |