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Method for asymmetrically amplifying multiple target nucleic acids

A target nucleic acid and nucleotide technology, which is applied in the field of asymmetric amplification of multiple target nucleic acids, can solve the problems of application limitations, inability to produce single-stranded products, etc., 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 multiple target nucleic acids
  • Method for asymmetrically amplifying multiple target nucleic acids
  • Method for asymmetrically amplifying multiple target nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0173] In this example, the HAND system, the common asymmetric PCR system and the system (primer set) of the present invention were investigated by taking the DNA fragment covering the gene polymorphism site rs8027171 on human chromosome 15 as the target nucleic acid to be amplified A situation where a single-stranded nucleic acid product is produced. The sequences of the primers and probes used in this example are shown in Table 1. The instrument used in this example is a SLAN 96 real-time fluorescent PCR instrument (Xiamen Zhishan Biotechnology Co., Ltd., Xiamen).

[0174] 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×Taq PCR buffer (TaKaRa, Beijing), 5.0 mM MgCl 2 , 0.2mM dNTPs, 1UTaq polymerase (TaKaRa, Beijing), 0.3μM rs8027171-P probe, 5μL human genomic DNA (the genotype of rs8027171 is G / A heterozygous) or negative control (water), and primers; wherein,

[0175] (1)...

Embodiment 2

[0187] In this example, the DNA fragment covering the gene polymorphism site rs8027171 on human chromosome 15 was used as the target nucleic acid to be amplified, and the influence of the ratio of the first and second universal primers on asymmetric amplification was investigated . The sequences of the primers and probes used in this example are shown in Table 1. The instrument used in this example is a SLAN 96 real-time fluorescent PCR instrument.

[0188] 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, 0.2mM dNTPs, 1U Taq DNA polymerase, 5μL human genomic DNA (the genotype of rs8027171 is G / A heterozygous), 0.02μM rs8027171-F, 0.02μM rs8027171-R, 0.3μM rs8027171-P probe, 0.06μM Tag 1 primer, and Tag 2 primer in the following amounts: 0.06 μM (Tag 1 / Tag 2 = 1 / 1), 0.24 μM (Tag 1 / Tag 2 = 1 / 4), 0.48 μM (Tag 1 / Tag 2 = 1 / 8), 0.72 μM (Tag 1 / Ta...

Embodiment 3

[0192] In this example, the genotyping of gene polymorphism sites rs48189298 and rs60871880 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 the primers and probes used in this example are shown in Table 2. The instrument used in this example is a SLAN 96 real-time fluorescent PCR instrument.

[0193] 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, 0.04 μM rs48189298-F, 0.04 μM rs48189298-R, 0.4 μM rs48189298-P, 0.06 μM rs60871880-F, 0.06 μM rs60871880-R, 0.4 μM Tag 10.8 rs3087 Primers, 1.6 μM Tag2 primer, 5 μL human genomic DNA or negative control (water). In this embodiment, two samples (sample 1 and 2) were detected, wherein the genotyp...

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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
CPCC12Q1/6844C12Q2537/143C12Q2531/107C12Q2527/107C12Q2563/107C12P19/34Y02A50/30C12Q1/6853C12Q2525/155C12Q2525/161C12Q2527/143C12Q2531/113C12Q1/6818C12Q1/6848C12Q1/686
Inventor 黄秋英李庆阁
Owner XIAMEN UNIV
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