Multiple PCR method based on selective probe and application thereof

Abstract

The present invention relates to nucleic acid amplification, and is especially multiple PCR process based on selective probe for simultaneous amplification of several nucleic acid segments and its application. The multiple PCR process includes the following steps: 1. constructing selective probe; 2. extension-coupling reaction; 3. PCR amplification; and 4. detecting PCR amplification product. The present invention features the selective probe and the PCR amplification product with difference in length. The process has greatly increased analysis flux, saving in the nucleic acid template consumption for detecting the sample, high compatibility of the detection system and other advantages.

Description

technical field [0001] The invention relates to nucleic acid amplification, in particular to a selective probe-based multiple PCR method and its application. Background technique [0002] Multiplex PCR (Polymerase Chain Reaction) technology is a technology for simultaneously amplifying multiple target nucleic acids in the same reaction tube, among which the most typical representative is a series of gene analysis technologies developed based on padlock probes. The classic padlock probe is composed of three parts, wherein two parts of the sequence are oligonucleotide fragments at both ends of the padlock probe, the two parts of the oligonucleotide fragments are complementary to the target nucleic acid, and the third part of the oligonucleotide fragment Some oligonucleotide fragments are connected into a whole. When the padlock probe forms a hybrid with the target nucleic acid, there is no space between the 5'- and 3'-ends of the padlock probe, and the two are closely adjacent...

AI Technical Summary

Problems solved by technology

Since CpG islands are CG-rich sequences, when analyzing DNA methylation based on MSRE, it is difficult to amplify multiple DNA sequences at the same time, even with multiplex PCR amplification based on padlock probes or molecular inversion probes. The large number of CpG sites in CpG leads to the existence of multiple MSRE sites. Therefore, according to the design scheme of classic padlock probes or molecular inversion probes, it is difficult to obtain probes that meet the requirements.

In addition, for the bisulfite-modified sequence, since the non-methylated cytosine has been converted into uracil, resulting in the simplicity of its sequence, the probes of the classic padlock probe or molecular inversion probe are also Difficulty getting probes that meet the requirements

[0004] At present, most of the multiplex PCR methods are limited to simultaneously amplifying 5 to 10 target nucleic acid fragments. The main reason is that n-fold PCR must add n pairs of different primers, that is, 2n primers, which makes the PCR amplification reaction much easier. Conditions are difficult to control, and in addition, primer-primer interactions are prone to primer dimerization and non-specific amplification

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