Design method for primers and probe for amplifying low-concentration mutation target sequence

Abstract

The invention discloses a design method for primers and a probe for amplifying a low-concentration mutation target sequence. The design method includes the steps that the mutation position (a mutational site, namely a 0 site) of a target sequence to be amplified is determined; a 15-25 bp nucleic acid fragment including the 0 site base is selected in the negative direction of the mutational site to serve as the forward primer, and a 12-25 bp nucleic acid sequence is selected from the 1 site base or the 0 site base in the positive direction of the mutational site to serve as a probe sequence of an amplification system; the reverse primer is designed at the suitable position of the 3'direction downstream of the probe sequence according to a conventional method. According to the design method for the primers and the probe, the target fragment can be effectively (with high specificity and efficiency) amplified under the background of a high-content wild type template particularly for point mutation, deletion mutation and insertion mutation in gene mutation, a fluorescence real-time PCR technology is combined, and the problem that at present, sensitivity is poor in clinic tumor detection and drug susceptibility detection can be solved effectively.

Description

technical field [0001] The invention belongs to the technical field of molecular biology and relates to a new method for designing nucleic acid primers and probes. More specifically, it relates to a method for designing primers and probes for amplifying low-concentration mutated target sequences. technical background [0002] Many diseases are closely related to gene mutations, and the type and generation of post-medication reactions in many cases are also related to gene mutations. The detection of gene mutations is of great significance in many fields and under certain circumstances, and the detection technology of gene mutations often uses PCR detection technology. PCR (Polymerase Chain Reaction) is the polymerase chain reaction, which refers to the DNA polymerase catalysis, using the parent chain DNA as a template, using a specific primer as the starting point of extension, and through steps such as denaturation, annealing, and extension, in vitro replication and parent...

AI Technical Summary

Problems solved by technology

When using PCR technology to detect gene mutations, especially when the amount of wild-type DNA accounts for a large proportion (that is, fewer mutations), the shortcomings of existing PCR technology are mainly reflected in the design of primers and probes, specifically as follows: ① specificity Poor selectivity, primers and probes designed according to common methods for gene mutations often produce non-specific amplification bands; The ability to detect mutant DNA is limited, and most of the tumor-causing mutations are somatic mutations, and mutant cells are mixed with wild-type cells, so the extracted DNA also contains a large amount of wild-type DNA;③ When the sample size is small, or there is complex background interference in the sample, it is difficult to use the extracted DNA to produce effective DNA amplification, and even lead to no detection, or false negative detection

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