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

A kit and multiple technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of complex detection methods, difficulty in popularization, cumbersome and other problems, and achieve the reduction of mutual interference probability, good economic benefits, and technical solutions bottleneck effect

Active Publication Date: 2012-02-15
北京凡知医学科技有限公司
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  • Claims
  • Application Information

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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

Method used

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

Examples

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Embodiment 1

[0030] The multiple PCR application technology system mediated by public primers, the amount of multiple primers in a 20 μL multiple PCR reaction system was changed from 10 12 Copy reduced to 10 4 Copy below, greatly reducing the interaction between multiple pairs of primers in the same reaction system; at the same time, because the interaction between multiple pairs of primers is greatly reduced, the reaction system can become an "open" reaction system, so that the increase in the same reaction system New detection genes become possible.

[0031] In this example, the feasibility and efficiency of the working principle of multiple primers combined with public primers were verified by using samples from patients clinically confirmed to carry the E2A / PBX1 fusion gene. The public primers were screened from the phage genome, and there was no specific product with human genome PCR amplification. See Table 1 for the sequences of public primers and multiple primers with public prim...

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PUM

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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...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 孙万平王伟大姚利陈燕李凯陈子兴
Owner 北京凡知医学科技有限公司
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