Method for rapidly detecting 3'-5' exo-activity of DNA polymerase or mismatching and kit thereof

Abstract

The invention relates to a method for detecting 3'-5' exo-activity of DNA polymerase or mismatching and a kit thereof. The method comprises the following steps: providing a nucleic acid of a known sequence, detecting a primer which is complementary to a nucleic acid with the known sequence, wherein the last deoxynucleotide at the 3' hydroxyl end and the first two deoxynucleotides to-be-polymerizedare different from each other; hybridizing the nucleic acid with the known sequence and a detection primer, and adding the DNA polymerase to be tested and the fluorescently labeled deoxynucleotide different from the deoxynucleotide to-be-polymerized for the first signal collection; adding exo-free Taq DNA polymerase and adding the first fluorescently labeled deoxynucleotide to-be-polymerized andthe second fluorescently labeled deoxynucleotide to-be-polymerized, and performing secondary signal collection; comprehensively analyzing a first signal and a second signal to determine whether the DNA polymerase to-be-tested is circumscribed or mismatched. The method can detect DNA polymerase 3'-5' exo-activity or mismatching when four nucleotides are present alone in a reaction system.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method and a kit for rapidly detecting DNA polymerase 3'-5' excision activity or mismatch. Background technique [0002] The use of dNTPs with free hydroxyl groups at the 3' end for sequencing can greatly increase the sequencing speed. New sequencing technologies such as single-molecule sequencing are more inclined to use this 3'-OH dNTP to achieve the goal of fast and long read length. However, when using this kind of dNTP, in many cases, four deoxynucleotides need to be added separately in a certain order, and only one kind is added at a time, which requires that the DNA polymerase used has only one kind of dNTP in the reaction system When present, it can faithfully add correctly paired dNTPs sequentially, without 3'-5' exoactivity or mismatch. In practical applications, it was found that when four dNTPs are present at the same time, there is no 3'-5' exoactivity ...

AI Technical Summary

Problems solved by technology

However, these methods cannot detect the exosome activity of DNA polymerase in the presence of a single dNTP that does not match the deoxynucleotide at the position to be tested.

[0005] Other 3'-5'exolytic activity detection methods are also limited to detecting the 3'-5'exolytic activity of DNA polymerase when the 3'end is mismatched, and cannot detect the activity of DNA polymerase when the 3'en

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