Suppression polymerase chain reaction kit

Abstract

The invention relates to a DNA in vitro amplification technology characterized by simplicity in operation and high specificity, namely a suppression polymerase chain reaction (S-PCR for short) kit. The process of the S-PCR comprises the following steps of: synthesizing a pair of special inhibition primers according to a target DNA sequence to be subjected to amplification, wherein about 15-18 basic groups at the 3' end of each primer are complementary with a template DNA sequence, and 11-14 basic groups at the 5' end of each primer are reversely complementary with the sequence of the primer; dissolving the primers to obtain a partial double-chain structure; and adding the inhibition primers into a PCR amplification buffer reaction system comprising heat-resistant DNA polymerase, dNTPs, template DNA, magnesium ions, PCR buffer, ultrapure water and the like, and performing high temperature, low temperature and intermediate temperature repeated thermal cycle by using a thermocycler. In the amplification process, when the temperature is reduced to annealing temperature, the inhibition primers are specifically combined with a template, and massive inhibition primers renature into double-stranded primers; and because of the priority of intra-strand renaturation, two ends of most non-specifically amplified single strands renature to form hairpin structures, the activity of becoming effective PCR templates is lost, and the amplification specificity is improved.

Description

technical field [0001] The invention relates to a polymerase chain reaction kit, more specifically, the invention relates to a high-specificity DNA in vitro amplification technique that is simple to operate and can improve the specificity of PCR amplification, that is, inhibits polymerase A chain reaction detection kit belongs to the field of DNA in vitro amplification technology in molecular biology detection technology. Background technique [0002] Among the existing DNA amplification techniques in vitro, the polymerase chain reaction is the most important and effective common technique. [0003] In conventional PCR, primer dimers and other non-specific amplifications are often encountered. The most likely time for primer dimers and other non-specific amplifications is after the reaction solution is mixed and before the temperature rises to the set temperature. , followed by the first few cycles of amplification, because the concentration of primers is the highest at thi...

AI Technical Summary

Problems solved by technology

[0003] In conventional PCR, primer dimers and other non-specific amplifications are often encountered. The most likely time for primer dimers and other non-specific amplifications is after the reaction solution is mixed and before the temperature rises to the set temperature. , followed by the first few cycles of amplification, because the concentration of primers is the highest at this time, which is most likely to cause non-specific amplification, and once non-specific templates including primer dimers are generated, because their efficiency is equivalent to that of specific amplification, and Competing substrates and enzymes often enter the plateau of amplification at about the same time as specific amplification, which not only makes it difficult to judge the amplification result, but also reduces the efficiency of specific amplification, especially when amplifying crude clinical samples , which often lead to false positive and false negative results in clinical genetic diagnosis

[0004] There are hot-start method, solid paraffin film method, antibody-enzyme method, etc. for improving the specificity of PCR amplification, but these methods can only partially achieve the purpose of reducing non-specific amplification, and once non-specific templates are produced, None of them can effectively inhibit the continuation of non-specific amplification