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Method for asymmetrically amplifying target nucleic acid

A technology for target nucleic acid and nucleotide, applied in the field of asymmetric amplification of target nucleic acid, can solve problems such as application limitation and inability to produce single-stranded products, and achieve the effect of improving specificity

Active Publication Date: 2021-06-29
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the PCR amplification using the HAND system is symmetrical and cannot produce single-stranded products, which limits the application of the HAND system in technical fields such as gene chips and probe melting curve analysis.

Method used

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  • Method for asymmetrically amplifying target nucleic acid
  • Method for asymmetrically amplifying target nucleic acid
  • Method for asymmetrically amplifying target nucleic acid

Examples

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Effect test

Embodiment 1

[0163] In this example, the DNA fragment covering the gene polymorphism site rs2252992 on human chromosome 21 was used as the target nucleic acid to be amplified, and the HAND system, the common asymmetric PCR system and the system (primer set) of the present invention were investigated to produce a single The case of strand nucleic acid products. The sequences of the primers and probes used in this example are shown in Table 1. The instrument used in this example is SLAN96 real-time fluorescent PCR instrument (Xiamen Zhishan Biotechnology Co., Ltd., Xiamen).

[0164] In short, in this example, a 25 μL PCR reaction system was used for PCR amplification and melting curve analysis. The PCR reaction system included: 1×TaqPCR buffer (TaKaRa, Beijing), 5.0 mM MgCl 2 , 0.2mM dNTPs, 1UTaq DNA polymerase (TaKaRa, Beijing), 0.2μM rs2252992-P probe, 5μL human genomic DNA (rs2252992 genotype is A / A homozygous) or negative control (water) and primers; wherein,

[0165] (1) Add 0.03 μM r...

Embodiment 2

[0177] In this example, the DNA fragment of the gene polymorphism site rs2252992 on human chromosome 21 was used as the target nucleic acid to be amplified, and the differences between the second universal sequence and the first universal sequence were investigated (that is, the second universal sequence The effect of different variant types of the sequence relative to the first universal sequence) on asymmetric amplification. The sequences of the primers and probes used in this embodiment are shown in Table 2, wherein the 3' terminal nucleotide of the universal primer (Tag primer) used is A; and 5 forward primers were designed, namely: The 3' terminal nucleotide of the second universal sequence of rs2252992-F-C is C, the 3' terminal nucleotide of the second universal sequence of rs2252992-F-G is G, and the 3' terminal nucleotide of the second universal sequence of rs2252992-F-T The acid is T, the 3' terminal nucleotide of the second universal sequence of rs2252992-F-A is A, a...

Embodiment 3

[0185] In this example, the genotyping of gene polymorphism sites rs2252992 and rs4816597 is taken as an example to illustrate that the system of the present invention can realize double and asymmetric amplification in a single reaction tube and be used for probe melting curve analysis. The sequences of primers and probes used in this example are shown in Table 3. The instrument used in this example is a SLAN 96 real-time fluorescent PCR instrument.

[0186] In short, in this example, a 25 μL PCR reaction system was used for PCR amplification and melting curve analysis, and the PCR reaction system included: 1×Taq PCR buffer, 5.0 mM MgCl 2 , 0.2 mM dNTPs, 1 U Taq DNA polymerase, 0.05 μM rs4816597-F, 0.05 μM rs4816597-R, 0.4 μM rs4816597-P, 0.04 μM rs2252992-F, 0.04 μM rs2252992-R, 0.4 μM rs2251.962-P Primers, 5 μL human genomic DNA or negative control (water). In this embodiment, four samples were detected (sample 1, 2, 3 and 4; each PCR reaction system detected a sample), wh...

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Abstract

The invention relates to multiplex, asymmetric amplification of nucleic acid molecules. Particularly, the invention provides a method for simultaneously and asymmetrically amplifying one or more target nucleic acids in a sample. The method can be used for simultaneously and asymmetrically amplifying multiple target nucleic acids in the sample and simultaneously generating a large number of single-chain products.

Description

technical field [0001] The present application relates to multiplexed, asymmetric amplification of nucleic acid molecules. In particular, the present application provides a method for simultaneously and asymmetrically amplifying one or more target nucleic acids in a sample. single-stranded product. Background technique [0002] Asymmetric PCR, first described by Gyllensten et al. (Proc.Natl.Acad.Sci.USA 1988, 85:7652-7656), refers to a method that uses unequal amounts of a pair of primers to generate large amounts of single-stranded DNA (ssDNA) . The single-stranded DNA produced by asymmetric PCR can be used for sequencing, as a probe, or to improve the detection signal in real-time PCR, gene chip detection, and probe melting curve analysis. However, traditional asymmetric PCR usually requires careful optimization to maximize the production of specific single-stranded products and minimize non-specific amplification. When multiple target nucleic acid sequences need to be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q2531/107C12Q2537/143C12Q2527/107Y02A50/30C12Q1/686C12Q1/6853C12Q2525/161C12Q2527/125C12Q2535/122C12Q2525/155C12Q2531/113C12Q2527/143C12Q1/6876C12Q2600/16
Inventor 黄秋英李庆阁
Owner XIAMEN UNIV
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