Direct use type high-fidelity PCR amplification reagent mixture

Abstract

The invention provides a direct use type high-fidelity PCR amplification reagent mixture which is high in fidelity, stability and using convenience. The reagent mixture comprises DNA polymerase, KCl, Tris-HCl, MgSO4, (NH4)2SO4, dNTPs and glycerinum, and ddH2O serves as the solvent. In certain optimized embodiments, the reagent mixture further comprises an optimizer which is one or more of DMSO, tetramethylene sulfoxide, hydroxyproline, trehalose, ethanediol and BSA. In use, PCR can be conducted simply by adding a template and a primer to an amplification system, operation processes are simplified greatly, operation time is shortened, and pollution is reduced; meanwhile, due to the fact that the system contains the optimizer, PCR amplification fidelity and stability can be improved remarkably, and the fidelity of the reagent mixture reaches 2.3*10<-7> to 3.9*10<-7>.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a direct-use high-fidelity PCR amplification mixed reagent. Background technique [0002] Polymerase Chain Reaction (Polymerase Chain Reaction, PCR) is a molecular biology technique used to amplify specific DNA fragments, and to some extent is a special DNA replication process outside the body. The biggest feature of PCR technology is to use a small amount of DNA template to achieve its massive enrichment. This technology was first conceived by Mullis of the United States in 1983. In 1985, he invented the polymerase chain reaction, that is, a simple DNA amplification method, which means the real birth of PCR technology. So far, PCR has been developed to the fourth generation technology. [0003] PCR is to use the denaturation of DNA molecules at a high temperature of about 95°C to become a single strand. At a low temperature of about 60°C, primers and single strands are ...

AI Technical Summary

Problems solved by technology

However, most of the commercially available direct-use PCR amplification reagents use TaqDNA polymerase enzyme as the polymerase, and the fidelity is about 10. -5 The fidelity of a few direct-use amplification reagents prepared by PfuDNA polymerase can reach 10 -6 about

In order to improve the fidelity of the polymerase, researchers or manufacturers have begun to try to add some additives to the PCR amplification reagents. Although these additives can improve the fidelity of the polymerase to a certain extent, they also affect the polymerase to a certain extent. The activity and stability of the direct-use PCR amplification reagents currently available on the market are not balanced due to the unbalanced addition of components, the stability of the entire system is poor, and the freeze-thaw resistance is relatively weak. After several freeze-thaws, the catalytic activity and fidelity of the polymerase are obvious. reduce

And the shelf life is short, the shelf life at room temperature is about one to two weeks, and the shelf life at 4°C is only about one month.

Unable to meet the requirements of fidelity, stability and convenience for PCR amplification reagents in practical applications

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