Method for isothermally amplifying nucleic acid fragment, primer set and application thereof, and kit used for amplifying nucleic acid fragment

Abstract

The invention relates to the field of biotechnology, and specifically relates to a method for isothermally amplifying a nucleic acid fragment, a primer set and an application thereof, and a kit used for amplifying the nucleic acid fragment. The method for isothermally amplifying the nucleic acid fragment specifically includes the following steps: employing the primer set comprising a specific primer pair and a universal primer pair, filling in an incision formed by combining the specific primer pair with an amplification template to make the universal primer pair utilize a filling-in product as a template, and isothermally amplifying a nucleic acid template under catalysis of DNA polymerase. The invention provides a new method for isothermally amplifying the nucleic acid fragment, and themethod is suitable for double-stranded DNA, single-stranded DNA and RNA amplification.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isothermally amplifying nucleic acid fragments, a primer set and its application, and a kit for amplifying nucleic acid fragments. Background technique [0002] Nucleic acid amplification is an essential reaction in living organisms, and the technology generated by its in vitro application has laid the foundation of modern molecular biology. One of the most commonly used techniques is the polymerase chain reaction (PCR), which requires a thermal cycler to some extent restricting the application of PCR. However, isothermal amplification does not require a thermal cycler. Researchers have developed many isothermal amplification methods based on the natural amplification process of nucleic acids. Existing methods can be divided into isothermal amplification methods based on RNA transcription, isothermal amplification methods based on DNA replication, The isothermal amplifi...

AI Technical Summary

Problems solved by technology

Disadvantages of NASBA: (1) It is not a true isothermal amplification reaction, which requires high temperature denaturation or annealing; (2) Due to the poor heat resistance of the reaction enzyme, it needs to be added separately; (3) Only the target sequence of 120-250bp can be effectively amplified

HDA has successfully amplified genomic DNA at a low temperature of 37°C. The disadvantages of HDA at present are the slow reaction speed, the poor continuous melting ability of E.coli UvrD helicase, and the lack of coordination between helicase and DNA polymerase.

The disadvantages of RCA are: the pretreatment is cumbersome, the reaction template is required to be single-stranded circular DNA, and samples that do not meet the requirements must be annealed and circularized; the reaction time is long, at least 4 hours

LAMP also has certain defects: due to the large number and concentration of primers required for the reaction, non-specific amplification is very easy to occur, resulting in false positives; the target sequence length can be effectively amplified only when the length of the target sequence is 200-300bp; contamination is very easy to occur during the operation and thus Generate false positives; LAMP also has great operational obstacles in terms of product recovery, identification, cloning, and single-strand isolation

However, due to the complexity of the reaction principle and reaction system, SDA has many disadvantages: (1) the use of non-standard nucleotides, the price of thio-modified single nucleotides is many times higher than that of ordinary single nucleotides, increasing (2) The mononucleotide modified by sulfur group is not the natural substrate of DNA polymerase, and the modified nucleotide competes with the standard nucleotide, and the polymerase is easy to drop from the DNA template, reducing the Therefore, SDA cannot synthesize longer products, generally no more than 200bp; (3) the end of the SDA product has a recognition sequence of a restriction endonuclease or its residue, which is not suitable for cloning directly; (4) ) The isothermal process of SDA is a two-step method, which requires a variable temperature process to open the double strand and anneal the primer. The catalytic enzyme is added after the variable temperature link, which increases the possibility of external contamination

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