Asymmetrical PCR amplification method, dedicated primer and use thereof

Abstract

The invention discloses an asymmetric PCR amplification method and its application, and aims to provide a simple and efficient PCR amplification method for preparing single-stranded amplification products. The asymmetric PCR primers provided by the present invention include several pairs of PCR primers, and are characterized in that: an oligonucleotide unrelated to the target sequence to be amplified is added to the 5' end of each pair of primers. acid tail. The asymmetric PCR amplification method provided comprises the following steps: 1) pre-denaturation; 2) the first part of PCR amplification including several cycles of denaturation, primer annealing and primer extension; 3) several cycles of denaturation, primer annealing and primer extension The second part of PCR amplification; the 5' end of one primer in the PCR primer pair used in the primer extension is added with an oligonucleotide tail that has nothing to do with the target sequence to be amplified. The asymmetric PCR amplification method of the present invention has a high single-strand yield, can perform single or multiple PCR amplification, and can be widely used in nucleic acid detection.

Description

Technical field [0001] The present invention relates to a PCR amplification method and its primers and applications, in particular to an asymmetric PCR amplification method, and the asymmetric PCR primer and its application in nucleic acid detection. Background technique [0002] With the continuous progress of life science research, people have realized that nucleic acid is the key material that determines genetic information. By detecting whether there is a certain nucleic acid(s) and its sequence variation in the sample, it can be judged whether the test object carries a certain pathogen. Microorganisms and their drug resistance, suffering from a certain disease or in a certain genetic state, etc. Therefore, nucleic acid analysis technology has been widely used in many fields of life science research, including identification, typing and drug resistance detection of pathogenic microorganisms; disease diagnosis and prognosis; HLA typing; SNP detection, etc. [0003] Since the a...

AI Technical Summary

Problems solved by technology

This method uses the purified symmetrical PCR product as a template, and uses a single primer to perform PCR cycles to prepare a single strand, but it must be completed in multiple steps, which is time-consuming and inconvenient.

[0017] At present, conventional multiplex PCR requires multiple rounds of optimization, and there are the following problems: 1) Due to the large number of primers, different primers may cause false positive amplification; The primer pair is dominantly amplified and the amplification efficiency of some primer pairs is very low; 3) The reproducibility of the results is poor

In this case, it is difficult to obtain satisfactory experimental results by performing asymmetric multiplex PCR amplification.

The aforementioned asymmetric PCR strategies are not effective when performing parallel analysis of multiple target molecules

Single-tube one-step asymmetric multiplex PCR has not been reported

Images