Multiplex polymerase chain reaction (PCR) amplification method and reagent kit

Abstract

The invention discloses a multiplex polymerase chain reaction (PCR) amplification method and a reagent kit for multiplex PCR amplification. The multiplex PCR amplification method comprises the step of designing public primers and multiplex primers with the public primers. The public primer design needs to follow two rules that: (1) under any optimized amplification conditions, no specific products are produced during the amplification of the public primers and templates to be measured; and (2) when the specific products are obtained through the amplification of the multiplex primers with the public primers, the public primers have the capability of amplifying the specific products, and then, a multiplex PCR system is configured for amplification. Because of the introduction of the public primers, the sequence specificity primer pair concentration in the reaction system is reduced by the level of more than four pairs, the possibility of the multiplex primers in the low concentration togenerate the mutual action in the same reaction system is greatly reduced, the problem of the technical bottle neck in the multiplex PCR of mutual interference of different primer pairs can be solved, and the precondition can also be created for adding new primer pairs and increasing the detection gene number.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a multiplex PCR amplification method and a kit for multiplex PCR amplification. Background technique [0002] Multiplex PCR (multiplex PCR), also known as multiple primer PCR or composite PCR, is a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments. Its reaction principle, reaction reagents and operation The process is the same as general PCR. [0003] In a multiplex PCR reaction, the following reagents are generally included: two or more primer pairs, dNTP, MgCl 2 , PCR buffer, template DNA, Taq DNA polymerase, and other adjuvants (DMSO, glycerol, BSA), usually in order to obtain better amplification results, the amount of reagents used, the reaction conditions of PCR amplification, such as extension Temperature, extension time, annealing time, annealing temperature, nu...

AI Technical Summary

Problems solved by technology

Since multiple pairs of primers are prone to interact in the same reaction system, such as the formation of hairpin structures, dimer structures, etc., thereby affecting the amplification efficiency, this is the main reason for dividing into eight tubes for detection, but it also leads to this problem. Such detection methods are complex and cumbersome, and have high requirements for operators related to the profession, so it is difficult to popularize

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