Method and reagent for constructing nucleic acid single-stranded circular library

Abstract

The invention discloses a method and reagents for constructing a nucleic acid single-strand circular library. The method comprises the following steps: using a transposase embedding complex to randomly interrupt the nucleic acid to form a first joint at both ends and a gap. Fragment; connect the second linker at the gap; perform the first PCR reaction to obtain the product of connecting the first linker and the second linker sequence at both ends; enzyme digestion to generate a nick, and then perform double-stranded circularization to generate a circular nucleic acid molecule; from Carry out restriction nick translation reaction at the nick; digest the part that has not occurred restriction nick translation reaction; connect the third adapter and oligonucleotide adapter sequence; perform the second PCR reaction to obtain the third adapter and oligonucleotide at both ends respectively The product of the acid linker sequence; isolate the single-stranded nucleic acid; circularize the single-stranded nucleic acid to obtain a single-stranded circular library. The method of the present invention realizes simple and rapid construction of a nucleic acid single-stranded circular library through the combination of transposase breaking and restriction gap translation reaction.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method and reagent for constructing a nucleic acid single-stranded circular library. Background technique [0002] Since Roche invented the pyrosequencing method and opened up the next-generation sequencing, until now, the next-generation sequencing has experienced a period of rapid development. However, with the development of high-throughput sequencing, high-throughput and low-cost sample preparation has gradually become a key consideration in the field of sequencing. Various principles of sample processing methods and automated devices have been continuously developed, mainly including sample fragmentation, terminal treatment of nucleic acid molecules, adapter ligation and final warehouse delivery. [0003] Among them, fragmentation is mainly divided into physical methods (such as ultrasonic shearing) or enzymatic methods (ie, non-specific endonuclease treatment) ...

AI Technical Summary

Problems solved by technology

[0004] From the perspective of the simplicity of various operations, the method of transposase interruption is undoubtedly far superior to other methods in terms of throughput and ease of operation, but this method of interruption also has its own disadvantages: transposase achieves transposition Depends on a specific 19bp Me sequence

Therefore, although transposases can add different adapter sequences to the 5' end and 3' end of the target sequence by embedding two completely different adapter sequences, the adapters all need to contain Me-specific sequences, and the consequence is to interrupt Both ends of the generated fragment will have a symmetrical Me sequence, and due to the special action of the transposase, there will be a 9nt gap between the target sequence and the Me sequence

The completely identical Me sequences adjacent to the target sequence will affect some downstream technical applications, such as the next-generation sequencing technology based on the ligation method. The Me sequences on both sides of the same chain are complementary sequences, which easily leads to single-stranded Annealing inside the molecule is not conducive to the binding of the anchor primer

[0005] So far, there is no patent or other literature reporting a molecular biology experiment method that can use transposase technology to break the target sequence extremely efficiently and quickly and correct the broken sequence into two completely different sequences

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