Oligonucleotide primer, reagent kit and application

Abstract

The invention belongs to the fields of a molecular biology detection technique and accurate medicines, and particularly relates to an oligonucleotide primer, a reagent kit and an application. The oligonucleotide primer comprises 3p arms at 3 'terminal and 5' terminal, a hairpin loop and 5p arms at the 3'terminal and the 5' terminal, wherein the 5p arms are not combined with a DNA template, the 3parms are hybridized with the DNA template, and besides, sequence specificity that polymerase extends is provided. The hairpin loop is located between the 5p arms and the 3p arms, and comprises a single-stranded loop hybridized with the DNA template and dual-stranded stems; and when the oligonucleotide primer is combined with the DNA template, binding energy between the single-stranded loop and theDNA template is greater than that of the dual-stranded stems. The invention further provides the reagent kit and an application thereof to multiplex PCR. The problems that a conventional primer is low in specificity, poor in homogeneity and high in detection sensitivity deviation, multiplex PCR primer dimers are formed, amplification preference is high, and mispairing rate is high, are solved, and the multiplex PCR effect is stable and reliable.

Description

technical field [0001] The invention belongs to the field of molecular biology detection technology and precision medicine, and specifically relates to an oligonucleotide primer, a kit and an application. Background technique [0002] With the development of high-throughput sequencing technology and the reduction of sequencing costs, its research and application in the fields of life sciences and medicine are becoming more and more extensive, especially in the diagnosis and prevention of diseases. Screening, tumor diagnosis, major disease prevention, health-related metagenomic analysis, etc. Although human whole genome sequencing has made a huge leap forward in terms of sequencing time and capital cost, the huge data analysis and genetic information extraction are time-consuming and laborious. High-coverage sequencing can also detect low-frequency mutations. Using amplicon sequencing, researchers can focus on key regions of interest in the genome. This highly targeted app...

AI Technical Summary

Problems solved by technology

[0007] Aiming at the fact that multiplex PCR in the prior art has amplification bias, amplification mutation and other factors that lead to uneven amplification of the target region, which has a biased impact on the subsequent sequencing data, and the Ω primers in the prior art have low specificity, poor uniformity and poor detection efficiency. The problem of large sensitivity deviation

This scheme provides an oligonucleotide primer, a kit and its corresponding method, which solves the technical problems of dimer formation, amplification preference and high mismatch rate in primers in the prior art, enabling multiplex PCR The effect is stable and reliable

Images