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Multiplex PCR (Polymerase Chain Reaction) method for detecting proximal SNP (Single Nucleotide Polymorphism)

A multiplex and site technology, applied in the field of SNP detection, can solve the problems of lagging amplification exponential growth period, reduced IC-type product quantity, and low IC-type specific product yield, so as to achieve wide application, improve detection efficiency, and ensure a balanced sexual effect

Active Publication Date: 2013-09-25
AGCU SCIENTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This effect is most pronounced in the early cycles of PCR when both pairs of ARMS primers (OP and IP) are bound to the template for amplification, resulting in amplification directed by the inner ARMS upstream (or downstream) primer (IP) Lag in exponential growth phase (especially for inner primers close to outer primers), relatively low yield of IC-type specific products
The decrease in the amount of IC-type products guided by the primers upstream (or downstream) of the inner ARMS makes it difficult to type the SNP2 locus

Method used

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  • Multiplex PCR (Polymerase Chain Reaction) method for detecting proximal SNP (Single Nucleotide Polymorphism)
  • Multiplex PCR (Polymerase Chain Reaction) method for detecting proximal SNP (Single Nucleotide Polymorphism)
  • Multiplex PCR (Polymerase Chain Reaction) method for detecting proximal SNP (Single Nucleotide Polymorphism)

Examples

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no. 1 example

[0037] Part of the first embodiment

[0038] The template DNA used in this example and the comparative example was extracted using a magnetic bead method DNA extraction kit (Changchun Bokun Biotechnology Co., Ltd.).

[0039] All primers for the first SNP site and the second SNP site, shared primers, and outer competitive non-amplification primers were purchased from Shanghai Sangon Bioengineering Co., Ltd.

[0040] Common primers are modified with FAM fluorescence at the 5' end.

[0041] The outer competitive non-amplification primer is modified with C3Spacer at the 3' end.

[0042] The reaction mixture Reaction Mix contains dNTP, Mg 2+ , buffer solution, etc., using conventional configurations, prepared by Wuxi Zhongde Meilian Biotechnology Co., Ltd. Electrophoretic analysis after PCR was carried out using an ABI3130 genetic analyzer (manufactured by ABI).

[0043] In the following examples and comparative examples, the detected SNP sites are two adjacent SNP sites of 197...

Embodiment 1

[0060] In this example, the same common upstream primer (CP1) as in the comparative example was used; two ARMS primers at the SNP1a site: a primer for amplifying T base (OP-T1) and a primer for amplifying A base (OP-T1) -A1); two ARMS primers for SNP2a site: a primer for amplifying G base (IP-G1), and a primer for amplifying C base (IP-C1).

[0061] The competitive non-amplification primer designed by increasing the ARMS primer targeting the SNP1a site is named CNEP1, and the sequence is (5'-3')GAAA ACCAGAACCTTACCACC- C3Spacer (SEQ NO.6), with a Tm of 55.6°C, 17 bp of the underlined part is a competition region, and the remaining 4 bp at the 5' end is an adjustment region, and the 3' end of the sequence is modified with C3Spacer. ARMS-PCR amplification as shown below was performed using the above primers.

[0062] In the embodiment, the Tm value of the competitive primer is 55.6°C, the Tm values ​​of the outer primers are 58.4°C and 55.7°C respectively, and the length of the...

Embodiment 2

[0097]In this example, the same common upstream primer (CP2) as in the comparative example was used; two ARMS primers at the SNP1b site: a primer for amplifying A base (OP-A2) and a primer for amplifying G base (OP-A2) -G2); Two ARMS primers for the SNP2b site: a primer for amplifying C base (IP-C2), and a primer for amplifying T base (IP-T2).

[0098] The competitive non-amplification primer designed by increasing the ARMS primer targeting the SNP1b site is named CNEP2, and the sequence is (5'-3')CAAGTGCACTTTC CAGTA CACTTAC -NH2C6 (SEQ NO.12), Tm is 58.2°C, 12 bp of the underlined part is a competition region, and the remaining 13 bp of the 5' end is an adjustment region, and the 3' end of the sequence is modified with -NH2C6. ARMS-PCR amplification as shown below was performed using the above primers.

[0099] In the embodiment, the Tm value of the competitive primer is 58.2°C, the Tm values ​​of the outer primers are 57.4°C and 61.8°C respectively, and the length of the c...

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Abstract

The invention discloses a multiplex PCR (Polymerase Chain Reaction) method for detecting proximal SNP (Single Nucleotide Polymorphism) loci and relates to a multiplex DNA amplification technology. The multiplex PCR method disclosed by the invention is characterized by using one common upstream primer, two inner SNPs loci ARMS primers, two outer SNPs locus ARMS primers and one outer competitive inextensible primer, wherein the outer competitive inextensible primer of which the 3' terminal is modified can be used for preventing polymerase extension, competes with the outer ARMS primer for a template locus, and eliminates the inner amplification inhibition phenomenon. The multiplex PCR method disclosed by the invention can be used for effectively detecting a plurality of proximal SNP loci and is applicable to a common multiplex PCR reaction, a multiplex fluorescence PCR reaction and a multiplex PCR reaction required by DNA hybridization.

Description

technical field [0001] The invention relates to a new method for detecting SNP, in particular to a design method for balancing the amplification between two adjacent SNP sites when detecting two adjacent SNP sites based on a multiplex PCR method, and its application in the field of nucleic acid detection. Background technique [0002] Polymerase chain reaction (PCR) is a molecular biology technique in which two oligonucleotides are used as primers to catalyze the amplification of a DNA fragment located between the two primers by a DNA polymerase. Since the technology was established in 1983, it has become the main body and key method in the current life science research and related fields because of its high sensitivity, high efficiency and high specificity. The technology itself is also constantly improving, and a series of related technologies have been developed, such as nested PCR, real-time quantitative PCR, differential display PCR, immune PCR, multiplex PCR, etc. Amo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 郑卫国卢文翔孟祥和卢青葛海鹏葛斌文薛佳郭育林
Owner AGCU SCIENTECH