A method for improving the specificity of polymerase chain reaction

Abstract

The invention discloses a PCR (polymerase chain reaction) specificity improving method. In the method, a sequence which is nonhomologous from a template and incapable of forming stable secondary structure is added to the end 5' of a primer pair; further, reaction specificity is improved by adding tail primer to the end 5' on the basis of compound PCR conditions. The compound PCR conditions include, (1) 5-10 cycles of normal PCR reaction condition and (2) two-step PCR condition based on combination of high-temperature annealing and extension. Compared with normal PCR primer, the tailed primer can improve PCR specificity effectively without influence to flexibility; meanwhile, working concentration of the tailed primer is equal to or even lower than that of common primer, thus, PCR specificity can be improved remarkably on the normal PCR condition, and can be further improved by a way of combining with the compound PCR conditions. The method is adaptable to various PCR such as normal PCR and nested PCR, and is of high application value.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a method of increasing the specificity of the polymerase chain reaction. Background technique [0002] Polymerase chain reaction (PCR) is a method widely used in biotechnology to efficiently amplify fragments of deoxyribonucleic acid (DNA). The basic principle of conventional PCR is: according to the known target DNA sequence to be amplified, design a pair of facing specific primers that are completely complementary to the two single strands of the target DNA sequence, and then template DNA, primers, DNA polymerase, 4 kinds of deoxygenated Nucleotides (dNTPs) are put into the reaction buffer, and then the target DNA fragment is amplified through the process of high temperature denaturation, low temperature renaturation, and medium temperature extension. In the high-temperature denaturation stage, the double-stranded DNA of the template is denatured and...

AI Technical Summary

Problems solved by technology

However, when the specificity of the target DNA sequence to be amplified is not high and there are many similar sequences in the template DNA, the 3' end of the primer may have multiple binding sites on the template DNA. In this case, PCR There will be non-specific amplification. When the non-specific amplification phenomenon is serious, it may even cause the target fragment to fail to amplify. At this time, the PCR reaction fails.

[0004] At present, the commonly used methods to improve the specificity of PCR reactions mainly include: (1) designing multiple primers to screen high-specificity primers, but when the specificity of the target sequence is poor, the effect of this scheme is not good; (2) improving the PCR reaction Annealing temperature: when the primer has multiple non-specific binding sites on the template, compared with the specific binding sites, most of the non-specific binding sites are only part of the complementary binding between the primer and the template, and its binding force is weak, Poor stability, so when the annealing temperature increases, the non-specific binding sites of the primers decrease, and the specificity of the PCR reaction increases; however, this method also has limitations. When the target sequence or primer specificity is poor, multiple The difference in the binding stability of the primer binding site is often not large, and increasing the annealing temperature cannot effectively improve the specificity of the PCR reaction; (3) Apply high-fidelity Taq enzyme: This method is to further improve the specificity of the PCR reaction when the specificity of the primer is acceptable. Reaction-specific method, so there are many non-specific amplifications that cannot be solved by this method

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