Method and kit for rapid detection of DNA polymerase 3'-5' exo-activity or mismatch

Abstract

Methods and kits for rapid detection of DNA polymerase 3'‑5' exonuclease activity or mismatches. The method includes: providing a nucleic acid of known sequence, and detection primers, which are complementary to the nucleic acid of known sequence, and the last deoxynucleotide at the 3' hydroxyl end and the first two deoxynucleotides to be polymerized are different from each other ;Hybridize the nucleic acid of known sequence and the detection primer, add the DNA polymerase to be tested and the deoxynucleotide with fluorescent label, which is different from the deoxynucleotide to be polymerized, for the first signal collection; Taq DNA polymerase, and add the first deoxynucleotide to be polymerized with fluorescent label and the second deoxynucleotide to be polymerized with fluorescent label, and carry out the second signal collection; comprehensive analysis first The secondary signal and the second signal are used to determine whether the DNA polymerase to be tested undergoes excision or mismatch. It can detect DNA polymerase 3'‑5' exoactivity or mismatch when four nucleotides exist alone in the reaction system.

Description

Method and kit for rapid detection of DNA polymerase 3'-5' exonucleation activity or mismatch technical field The present invention relates to molecular biology technical field, be specifically related to a kind of rapid detection DNA polymerase 3 '-5 ' exo-cutting activity Methods and kits for sex or mismatch. Background technique [0002] The dNTP with a free hydroxyl group at the 3' end is used in sequencing, which can greatly improve the sequencing speed. New sequencing technologies such as single Molecular sequencing, etc. prefer to use this 3'-OH dNTP to achieve the goal of fast and long read length. But using this In the case of dNTP, in many cases, four deoxynucleotides need to be added separately in a certain order, and only one is added at a time. This requires that the DNA polymerase used can faithfully add the correct sequence when only one dNTP exists in the reaction system. Paired dNTPs must not have 3'-5' exo-activity or mismatch. It is found in pra...

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'end is paired correctly. 3'-5' exo activity

Therefore, it cannot be applied to the detection and screening of exonuclease activity of some sequencing enzymes

The traditional detection methods of DNA polymerase mismatch rate (such as blue-white spot test, plasmid endo-cutting test) are cumbersome to operate, and cannot be applied to the detection and screening of sequencing enzymes with special needs

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