CN1514006A - PCR method of universal primer and its reaction liquid and application in multiple PCR - Google Patents
PCR method of universal primer and its reaction liquid and application in multiple PCR Download PDFInfo
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Abstract
A PCR method for universal primer, its reaction liquid, and its application in multiple PCR are disclosed. Said PCR method features that a pair of leader primers complementary with template in region 3' and a pair of universal primers the same as said leader primer in region 5' are amplified in a single PCR. Its advantages are high reaction specificity, stable amplifying efficiency and wide concentration range of primer.
Description
Technical field
The present invention relates to Protocols in Molecular Biology, particularly relate to a kind of method and reaction solution and the application in multiplex PCR of polymerase chain reaction.
Technical background
PCR is a kind of simple, single-minded, sensitive, gene amplification method fast, and its process generally includes 20-40 circulation that is made of sex change, annealing and extension three-step reaction.The key of PCR method according to target gene order design a pair of have high preciseness, with two chains of target gene DNA complementary primers respectively.Double-stranded DNA is separated under denaturation temperature and is split into two strands, when reducing to annealing temperature, positive and negative primer respectively with two chain combinations of template DNA, archaeal dna polymerase holds the direction that begins by 5 ' to 3 ' to make chain extension at primer and template bonded 3 ' under elongating temperature then, thereby finishes a round-robin amplification.So using a pair of primer is the foundation stone of PCR method.From mid-1980s PCR invention up to now, though various improvement and deriving method emerge in an endless stream, but never break through above-mentioned basic model, the amplification procedure that is each target gene is all from start to finish by finishing with a pair of positive and negative primer of template complementary, and polymerase chain reaction method of the present invention has then adopted a kind of new pattern.
PCR method of the present invention has comprised the leading primer of a pair of plaing " primer of primer " effect and the omnipotent primer of a pair of energy widespread usage.A pair of leading primer only plays a role in junior three circulation of PCR reaction, and all the other of PCR reaction circulate and just finished separately by a pair of omnipotent primer.According to the principle of pcr amplification, target amplification product real synthetic is that the 3rd circulation from PCR begins later on strictly speaking, says on the meaning that from then on the amplification procedure of polymerase chain reaction of the present invention is realized by omnipotent primer from start to finish.
Existing multiplex PCR is to adopt many primer that is complementary with the To Template base sequence to be realized multiplex amplification to adding simultaneously in the PCR reaction tubes, all the time utilize and primary template complementary primer in the whole pcr amplification process, in fact, every pair of primer extends then with the template pairing separately, each PCR is unrelated, because primer self structure, and template joint efficiency and primer concentration the omnidistance variation of reaction each to primer between differ greatly, the amplification efficiency of each PCR also can't be consistent, even can produce the defective that some " weak tendency PCR " quilt " surging PCR " is covered, can't produce enough products.
Summary of the invention
Invent the technical problem of required solution
The technical problem of solution required for the present invention provides a kind of polymerase chain reaction method and reaction solution and application in multiplex PCR of omnipotent primer, to break through the routine of utilizing all the time in the whole pcr amplification process in the prior art with primary template complementary primer, the amplification efficiency that overcomes multiplex PCR is subjected to primer concentration, primer self structure and influences bigger defective with the template joint efficiency.
Technical scheme
Omnipotent primer order of the present invention and target gene sequence independence, therefore omnipotent primer design is totally independent of by cls gene, and each has designed definite omnipotent primer can be applicable to other range genes of amplification.
Omnipotent primer length is the 15-40 base, preferred length 18-30 base; Omnipotent primer calculates with the most contiguous base method, and melting temperature(Tm) is 60-100 ℃, and preferable range is 80-95 ℃; The hair clip handle base number of omnipotent primer should be low as far as possible, and preferred hair clip handle base number is 0, does not promptly have the oligonucleotide of hairpin structure; Omnipotent primer self 3 ' end complementary base radix should be low as far as possible, and preferred 3 ' to hold self complementary base radix be zero oligonucleotide; The total complementary base radix of omnipotent primer self should be low as far as possible, and preferably self total complementary base is zero oligonucleotide.
Positive and negative leading primer is partly formed by two, and 3 ' zone of leading primer is complementary fully with the target gene order, and available primer software or manual method design, and should meet the high preciseness of common outstanding primer.The length in leading primer 3 ' zone is the 15-40 base, preferred 20-30 base.5 ' the end order of positive and negative leading primer is all identical with omnipotent primer order.The total length of positive and negative leading primer is greater than 35 bases, preferred 40-50 base; The most contiguous base method melting temperature(Tm) of leading primer is 75-100 ℃, preferred 85-95 ℃, at above-mentioned omnipotent primer, 3 ' end of leading primer should avoid matching between more self pairing or positive anti-primer base, particularly do not have three same bases of above successive, avoid occurring hairpin structure and cause producing primer dimer.
Leading primer and omnipotent primer are mixed together with other reagent when assembling PCR reaction solution, because omnipotent primer order does not match with target gene, do not play effect fully in the first few of reaction circulates.5 ' end of leading primer has only a few base and template matches, but its 3 ' end mates fully with template and higher binding strength is arranged, so still can anneal with target gene under higher temperature single-mindedly.Through three round-robin reactions, in reaction solution, formed complete target amplification product, 5 ' the end order of every chain of amplified production is complementary fully with the order of omnipotent primer, so omnipotent primer can play a role in circulation subsequently.
The reaction solution of omnipotent primer PCR reaction among the present invention, its composition comprises: a pair of leading primer and a pair of omnipotent primer primer, four kinds of thymus nucleic acids, magnesium ion, damping fluid, hot resistant DNA polymerase and target dnas.Usually the primer ultimate density of PCR reaction is at 100-1000nM, the function of leading primer is just supported first few round-robin amplified reaction among the present invention, ultimate density in reaction solution is 5-100nM, preferred 5-50nM, and half that is common primer concentration lower limit is to 1/20th.And the ultimate density 1-500 μ M of omnipotent primer in reaction solution, preferred 10-100 μ M.In other words, the high 100-10 of the leading primer of the concentration ratio of omnipotent primer in the PCR reaction solution, 000 times, the melting temperature(Tm) of omnipotent primer and leading primer are approaching, so, just in amplification procedure, play a leading role when PCR enters the omnipotent primer of the 4th circulation, finish until reaction with the alternative guide's primer of its concentration advantage.
The polymerase chain reaction of said omnipotent primer also is adapted at using in the multiplex PCR, the difference be to add simultaneously in the reaction system many to leading primer of different templates sequence complementary and a pair of omnipotent primer, the remaining reaction condition is identical.Leading primer causes a plurality of reproduction processes simultaneously, after three round-robin reactions, form a plurality of complete target amplification products in reaction solution, 5 ' the end order of every chain of amplified production is complementary fully with the order of omnipotent primer, and omnipotent primer can play a role in circulation subsequently.
Template DNA of the present invention is cDNA, utilize cDNA to prepare test kit (Advantage-RT-for-PCR-kit by the total RNA of people's muscle tissue (1 μ g/1 λ, Clontech company), Clontech company) preparation, the Oligo that the reverse transcription the primer provides for test kit (dT)
18, PCR reaction volume 10 μ L, every pipe adds cDNA, 1 μ L.The Advantage of Clontech company 2 test kits are all adopted in PCR reaction of the present invention.
Beneficial effect
Omnipotent primer PCR method has an enormous advantage than standard pcr.The first, leading primer concentration than the low 10-100 of common primer concentration doubly improves the preciseness of reaction, helps reducing or eliminating non-single-minded amplified production and primer dipolymer.The second, the main process of amplification is finished by omnipotent primer, has avoided common melting temperature(Tm) owing to positive and negative primer, bonding strength, concentration and not quite identical with the amplification efficiency of not equal two chains that cause of template bonding strength.Promptly eliminated the possible negative impact of inhomogeneity to increasing between the primer.The 3rd, omnipotent primer is independent of gene order in proper order and designs, and has the ideal preciseness, final concentration is high and do not have a negative impact in the reaction solution, and the appearance of high concentration universal primer deferrable PCR flat slope, the straight line accumulated phase of expansion product helps the realization of multiplex PCR or multiple quantitative PCR.
The multiple PCR method of omnipotent primer is compared with common multiplex PCR, owing to manyly leading primer caused each each amplified production of self-template synthetic in the circulation of the overwhelming majority of PCR whole process, enjoy with a pair of omnipotent primer, and react with same efficiency, so make eliminated between each reaction primer concentration, primer self structure and with the difference of template joint efficiency, multi-PRC reaction is carried out under the background of unanimity, improve the accuracy that detects, avoided occurring false negative or the incorrect sxemiquantitative judgement that the gene-amplification efficiency variance causes.
Embodiment
Embodiment 1.
Omnipotent primer design: the principle according to omnipotent design of primers in the technical scheme designs following primer (table 1).
The omnipotent design of primers of table 1. for example
| Omnipotent primer U1 | Omnipotent primer U2 | |
| Length | 18 bases | 25 bases |
| Sequence (5 '-3 ') | ????CCCCC?CCCCC ????GCCCC?CCC | ??CCCCC?CCCCC ??CCTCC?CCCCC ??CTTTT |
| %GC method melting temperature(Tm) Tm (℃) | ????90.9 | ??90.5 |
| The most contiguous base method melting temperature(Tm) Td (℃) | ????86.6 | ??91.2 |
| Bonding strength | ????619 | ??639 |
| Hair clip handle base number | ????1 | ??0 |
| 3 ' end complementary base radix | ????0 | ??0 |
| The most stable total complementary base radix | ????2 | ??0 |
Embodiment 2.
Polymerase chain reaction based on omnipotent primer.
Design leading primer (table 2) at people actomyosin and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene order.
Table 2. with people actomyosin and G3PDH gene order for example designs leading primer (5 '-3 ')
| ?8A1U1(5′) | CCC?CCC?CCC?CGC?CCC?CCC?GCT?ACG?TCG?CCC?TGG?ACT?TC |
| ?8A2U1(3′) | CCC?CCC?CCC?CGC?CCC?CCC?CCG?CCA?GAC?AGC?ACT?GTG?TT |
| ?8G1U1(5′) | CCC?CCC?CCC?CGC?CCC?CCC?CGA?CAG?TCA?GCC?GCA?TCT?TC |
| ?8G2U1(3′) | CCC?CCC?CCC?CGC?CCC?CCC?ACG?TAC?TCA?GCG?CCA?GCA?TC |
| ?8G3U2(5′) | CCC?CCC?CCC?CCC?TCC?CCC?CCC?TTT?TAT?GGC?ACC?GTC?AAG ??????????????????????GCT?GA |
| ?8G4U2(3′) | CCC?CCC?CCC?CCC?TCC?CCC?CCC?TTT?TAG?TGA?TGG?CAT?GGA ????????????????????CTG?TGG?TC |
Primer concentration (μ M) in each omnipotent primer PCR reaction of table 3.
| The reaction sequence number | ??F1 | ??F2 | ??F3 | ??F4 | ??F5 | ??F6 | ??F7 | ??F8 | ??F9 | ??F10 |
| ??8A1U1 ??8A2U1 | ?0.005 | ??0.01 | ?0.005 | ??0.1 | ||||||
| ??U1 | ?1 | ??1?0 | ?100 | ??100 | ||||||
| ??8G1U1 ??8G2U1 | ??0.01 | 0.005 | ??0.1 |
| ?U1 | ????20 | ????100 | ????50 | |||||||
| ?8G3U2 ?8G4U2 | ?0.02 | ?0.05 | ?0.1 | |||||||
| ?U2 | ?1 | ?10 | ?100 |
Reaction conditions: above-mentioned leading primer and the concentration of omnipotent primer in reaction solution are as shown in table 3.PCR:95 ℃ 30 seconds, 68 ℃ 30 seconds, 72 ℃ 60 seconds, totally 25 circulations.
Omnipotent primer PCR result: above-mentioned F1-F10 is that template increases and all obtained positive findings with the complementary DNA that is obtained by people's total tissue RNA, and positive fragment length is respectively F1--F4 292bp, F5--F7 377bp, F8--F10 427bp.
Embodiment 3.
Application in multiplex PCR.
Add two couples of leading primer 8A1U1 (5 ')/8A2U1 (3 ') and 8G1U1 (5 ')/8G2U1 (5 ') in a PCR reaction tubes simultaneously, concentration is 0.01uM.And to add omnipotent primer 8U1 concentration be 5uM, and other reaction conditionss are with embodiment 2, and the result obtains two amplified bands, is respectively 292 and 377bp.
Nucleotide sequence
People actomyosin gene order
1?accgcgtccg?ccccgcgagc?acagagcctc?gcctttgccg?atccgccgcc?cgtccacacc
61?cgccgccagc?tcaccatgga?tgatgatatc?gccgcgctcg?tcgtcgacaa?cggctccggc
121?atgtgcaagg?ccggcttcgc?gggcgacgat?gccccccggg?ccgtcttccc?ctccatcgtg
181?gggcgcccca?ggcaccaggg?cgtgatggtg?ggcatgggtc?agaaggattc?ctatgtgggc
241?gacgaggccc?agagcaagag?aggcatcctc?accctgaagt?accccatcga?gcacggcatc
301?gtcaccaact?gggacgacat?ggagaaaatc?tggcaccaca?ccttctacaa?tgagctgcgt
361?gtgcctcccg?aggagcaccc?cgtgctgctg?accgaggccc?ccctgaaccc?caaggccaac
421?cgcgagaaga?tgacccagat?catgtttgag?accttcaaca?ccccagccat?gtacgttgct
481?atccaggctg?tgctatccct?gtacgcctct?ggccgtacca?ctggcatcgt?gatggactcc
541?ggtgacgggg?tcacccacac?tgtgcccatc?tacgaggggt?atgccctccc?ccatgccatc
601?ctgcgtctgg?acctggctgg?ccgggacctg?actgactacc?tcatgaagat?cctcaccgag
661?cgcggctaca?gcttcaccac?cacggccgag?cgggaaatcg?tgcgtgacat?taaggagaag
721?ctgtgctacg?tcgccctgga?cttcgagcaa?gagatggcca?cggctgcttc?cagctcctcc
781?ctggagaaga?gctacgagct?gcctgacggc?caggtcatca?ccattggcaa?tgagcggttc
841?cgctgccctg?aggcactctt?ccagccttcc?ttcctgggca?tggagtcctg?tggcatccac
901?gaaactacct?tcaactccat?catgaagtgt?gacgtggaca?tccgcaaaga?cctgtacgcc
961?aacacagtgc?tgtctggcgg?caccaccatg?taccctggca?ttgccgacag?gatgcagaag
1021?gagatcactg?ccctggcacc?cagcacaatg?aagatcaaga?tcattgctcc?tcctgagcgc
1081?aagtactccg?tgtggatcgg?cggctccatc?ctggcctcgc?tgtccacctt?ccagcagatg
1141?tggatcagca?agcaggagta?tgacgagtcc?ggcccctcca?tcgtccaccg?caaatgcttc
1201?taggcggact?atgacttagt?tgcgttacac?cctttcttga?caaaacctaa?cttgcgcaga
1261?aaacaagatg?agattggcat?ggctttattt?gttttttttg?ttttgttttg?gttttttttt
1321?tttttttggc?ttgactcagg?atttaaaaac?tggaacggtg?aaggtgacag?cagtcggttg
1381?gagcgagcat?cccccaaagt?tcacaatgtg?gccgaggact?ttgattgcac?attgttgttt
1441?ttttaatagt?cattccaaat?atgagatgca?ttgttacagg?aagtcccttg?ccatcctaaa
1501?agccacccca?cttctctcta?aggagaatgg?cccagtcctc?tcccaagtcc?acacagggga
1561?ggtgatagca?ttgctttcgt?gtaaattatg?taatgcaaaa?tttttttaat?cttcgcctta
1621?atactttttt?attttgtttt?attttgaatg?atgagccttc?gtgccccccc?ttcccccttt
1681?tttgtccccc?aacttgagat?gtatgaaggc?ttttggtctc?cctgggagtg?ggtggaggca
1741?gccagggctt?acctgtacac?tgacttgaga?ccagttgaat?aaaagtgcac?accttaaaaa
1801?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?a
People's glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene order
1??ctctctgctc?ctcctgttcg?acagtcagcc?gcatcttctt?ttgcgtcgcc?agccgagcca
61??catcgctcag?acaccatggg?gaaggtgaag?gtcggagtca?acggatttgg?tcgtattggg
121??cgcctggtca?ccagggctgc?ttttaactct?ggtaaagtgg?atattgttgc?catcaatgac
181??cccttcattg?acctcaacta?catggtttac?atgttccaat?atgattccac?ccatggcaaa
241??ttccatggca?ccgtcaaggc?tgagaacggg?aagcttgtca?tcaatggaaa?tcccatcacc
301??atcttccagg?agcgagatcc?ctccaaaatc?aagtggggcg?atgctggcgc?tgagtacgtc
361??gtggagtcca?ctggcgtctt?caccaccatg?gagaaggctg?gggctcattt?gcagggggga
421??gccaaaaggg?tcatcatctc?tgccccctct?gctgatgccc?ccatgttcgt?catgggtgtg
481??aaccatgaga?agtatgacaa?cagcctcaag?atcatcagca?atgcctcctg?caccaccaac
541??tgcttagcac?ccctggccaa?ggtcatccat?gacaactttg?gtatcgtgga?aggactcatg
601??accacagtcc?atgccatcac?tgccacccag?aagactgtgg?atggcccctc?cgggaaactg
661??tggcgtgatg?gccgcggggc?tctccagaac?atcatccctg?cctctactgg?cgctgccaag
721??gctgtgggca?aggtcatccc?tgagctgaac?gggaagctca?ctggcatggc?cttccgtgtc
781??cccactgcca?acgtgtcagt?ggtggacctg?acctgccgtc?tagaaaaacc?tgccaaatat
841??gatgacatca?agaaggtggt?gaagcaggcg?tcggagggcc?ccctcaaggg?catcctgggc
901??tacactgagc?accaggtggt?ctcctctgac?ttcaacagcg?acacccactc?ctccaccttt
961??gacgctgggg?ctggcattgc?cctcaacgac?cactttgtca?agctcatttc?ctggtatgac
1021?aacgaatttg?gctacagcaa?cagggtggtg?gacctcatgg?cccacatggc?ctccaaggag
1081?taagacccct?ggaccaccag?ccccagcaag?agcacaagag?gaagagagag?accctcactg
1141?ctggggagtc?cctgccacac?tcagtccccc?accacactga?atctcccctc?ctcacagttg
1201?ccatgtagac?cccttgaaga?ggggaggggc?ctagggagcc?gcaccttgtc?atgtaccatc
1261?aataaagtac?cctgtgctca?acc
Claims (10)
1. the polymerase chain reaction method of an omnipotent primer in turn includes the following steps:
(1) template sex change;
(2) primer annealing;
(3) synthetic complementary dna chain is extended in archaeal dna polymerase catalysis down;
(4) set by step (1)-(3) circulation carrying out amplified reaction;
It is characterized in that the primer comprises: a pair of 3 ' zone and the leading primer of template complementary, with the identical omnipotent primer in a pair of and leading primer 5 ' zone.
2. polymerase chain reaction method according to claim 1 is characterized in that the positive and negative primer sequence of a pair of omnipotent primer is identical or inequality.
3. polymerase chain reaction method according to claim 1 is characterized in that the length in leading primer 3 ' zone is that 15-40 base, melting temperature(Tm) are 75-100 ℃.
4. polymerase chain reaction method according to claim 1 is characterized in that the length in leading primer 3 ' zone is that 20-30 base, melting temperature(Tm) are 85-95 ℃.
5. according to each described polymerase chain reaction method in the claim 1,2 or 3, it is characterized in that omnipotent primer length is that 15-35 base, melting temperature(Tm) are 60-100 ℃.
6. according to each described polymerase chain reaction method in the claim 1,2 or 3, it is characterized in that omnipotent primer length is that 18-30 base, melting temperature(Tm) are 80-95 ℃.
7. the reaction solution of the described omnipotent primer-oligomerization polymerase chain reaction of claim 1 is characterized in that leading primer concentration is 5nM-100nM, and omnipotent primer concentration is 1 μ M-500 μ M.
8. the reaction solution of omnipotent primer-oligomerization polymerase chain reaction according to claim 11, composition comprises: a pair of leading primer and a pair of omnipotent primer primer, four kinds of thymus nucleic acids, magnesium ion, damping fluid, hot resistant DNA polymerase and target dnas, it is characterized in that leading primer concentration respectively is 5nM-100nM, omnipotent primer concentration respectively is 1 μ M-500 μ M.
9. the application of the polymerase chain reaction of the described omnipotent primer of claim 1 in multiplex PCR, it is characterized in that the reaction comprise many to leading primer of different templates sequence complementary and a pair of omnipotent primer.
10. the application of polymerase chain reaction according to claim 9 is characterized in that the positive and negative primer sequence of a pair of omnipotent primer is identical or inequality.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101899438A (en) * | 2010-04-16 | 2010-12-01 | 陕西北美基因股份有限公司 | Multiplex PCR primer for amplifying human EGFR gene and design method thereof |
| CN105483117A (en) * | 2015-12-11 | 2016-04-13 | 广州医科大学附属肿瘤医院 | PCR (polymerase chain reaction) specificity improving method |
| CN108998551A (en) * | 2018-07-23 | 2018-12-14 | 湖南省茶叶研究所（湖南省茶叶检测中心） | Tea tree quadruple fluorescence SSR molecular marker detection method |
| CN113832147A (en) * | 2021-09-08 | 2021-12-24 | 华南农业大学 | Efficient PCR primer for large-fragment DNA synthesis and amplification, method and application |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1316034C (en) * | 2001-03-28 | 2007-05-16 | 科学与工业研究会 | Universal primers for wildlife identification |
| CN1341749A (en) * | 2001-10-04 | 2002-03-27 | 吴昌 | Nucleic acid target molecule fragment selection and amplification method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899438A (en) * | 2010-04-16 | 2010-12-01 | 陕西北美基因股份有限公司 | Multiplex PCR primer for amplifying human EGFR gene and design method thereof |
| CN101899438B (en) * | 2010-04-16 | 2013-03-06 | 陕西北美基因股份有限公司 | Multiplex PCR primer for amplifying human EGFR gene and design method thereof |
| CN105483117A (en) * | 2015-12-11 | 2016-04-13 | 广州医科大学附属肿瘤医院 | PCR (polymerase chain reaction) specificity improving method |
| CN105483117B (en) * | 2015-12-11 | 2019-04-30 | 广州医科大学附属肿瘤医院 | A method of improving polymerase chain reaction specificity |
| CN108998551A (en) * | 2018-07-23 | 2018-12-14 | 湖南省茶叶研究所（湖南省茶叶检测中心） | Tea tree quadruple fluorescence SSR molecular marker detection method |
| CN113832147A (en) * | 2021-09-08 | 2021-12-24 | 华南农业大学 | Efficient PCR primer for large-fragment DNA synthesis and amplification, method and application |
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