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Method for performing multiplex PCR by utilizing hairpin primers

A hairpin, multiplex technology, applied in the field of multiplex PCR technology, can solve the problem of uneven target products, and achieve the effects of improving efficiency and throughput, increasing uniformity, and improving capture efficiency.

Active Publication Date: 2017-01-04
上海翼和应用生物技术有限公司
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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a method for multiplex PCR using hairpin primers, improve the throughput of multiplex PCR, and overcome the mismatch of target products caused by multiple pairs of primers and non-specific amplification in traditional multiplex PCR technology. uniform defect

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  • Method for performing multiplex PCR by utilizing hairpin primers
  • Method for performing multiplex PCR by utilizing hairpin primers
  • Method for performing multiplex PCR by utilizing hairpin primers

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Embodiment 1

[0033] Ion torrent PGM platform was used to detect single nucleotide polymorphism (single nucleotide polymorphism, SNP).

[0034] Sequence search and primer design: According to the SNP database of NCBI, a total of 89 target segment sequences carrying SNP sites in the rat genome were found, and Primer3 online software was used to design primers according to general rules, and the upstream and downstream of the designed primers were artificially inserted. Add the upstream and downstream universal sequences to the 5' end respectively, and add a sequence that is completely complementary to the 3' end of the target segment at the 5' end of the universal sequence to form a completely closed hairpin primer at the end, while ensuring that the hairpin primer melts The temperature was not lower than 80°C, and then primer synthesis was carried out (Table 1).

[0035] Table 1. SNP detection specific hairpin primers

[0036]

[0037]

[0038]

[0039]

[0040]

[0041] DNA...

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Abstract

The invention relates to a method for performing multiplex PCR by utilizing hairpin primers and a kit as well as application thereof to the establishment of a second-generation sequencing platform library. A specific sequence at the 3'end of each of the hairpin primers is completely paired with a target segment, the melting temperature is 80 DEG C or higher, and a hairpin structure is only opened in the degeneration process when the PCR is performed. The non-specific amplification and the primer dimer formation can be effectively reduced, so that the uniformity of multiple PCR products is improved. The method and the kit as well as the application thereof provided by the invention have the benefits that the two rounds of reaction is performed by utilizing the multiple hairpin primers, so that the second-generation sequencing library is quickly and conveniently established; different templates are distinguished through joint primers carrying different bar code sequences, and the different joint primers carry a same universal amplification primer, so that the difference among different template amplification products is reduced.

Description

technical field [0001] The invention relates to the field of nucleic acid detection, in particular to a multiplex PCR technique that utilizes hairpin primers to perform multiplex PCR, enriches the DNA sequences of target segments of multiple samples, and reduces primer dimers and non-specific amplification. Background technique [0002] Due to the low price and easy operation, multiplex PCR is an important means of target segment enrichment technology. Faced with the capture of target segments in a large number of complex genomic samples, multiplex PCR technology has become the preferred technology due to its advantages of strong specificity, low price, and good repeatability. At the same time, commercial multiplex PCR kits and non-commercial multiplex PCR techniques continue to emerge. According to the literature "Target-enrichment strategies for next-generation sequencing" (Naturemethods, 2010, 2010, 7(2): 111-118) and "Genotyping‐in‐Thousands by sequencing (GT‐seq): A co...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C40B50/06
CPCC12Q1/686C40B50/06C12Q2537/143C12Q2527/101C12Q2525/301
Inventor 肖君华陈科陈轶群
Owner 上海翼和应用生物技术有限公司
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