A method for multi-SNP identification based on high-resolution melting curves

Abstract

The invention belongs to the technical fields of the agriculture and the biology, and concretely discloses a high resolution melting curve-based multi-SNP identification method. The high resolution melting curve-based multi-SNP identification method is provided by combining multi-PCR, nested PCR and allele specific PCR against the limitations of present SNP identification. The utilization of the multi-PCR realizes 2-4 gene fragment amplification, improves the experiment efficiency, and reduces the experiment cost; the utilization of the nested PCR improves the specificity of amplified fragments, obtains a high quality and uniform concentration DNA template, and improves the stability of an HRM technology; the utilization of the allele specific PCR realizes SNP site discrimination, avoids fluorescent marker set or complicated electrophoresis and enzyme cutting operation; and the method comprehensively utilizing the multi-PCR, nested PCR and allele specific PCR realizes multi-SNP batch identification reduces the experiment cost.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a multi-SNP identification method based on a high-resolution melting curve. Background technique [0002] Single nucleotide polymorphisms (SNPs) refer to the polymorphisms caused by the substitution of single nucleotides (A, G, C, T) in the genomic DNA sequence, and are a new generation of polymorphic genetic markers. SNPs are widely present in biological genomes. For example, there are more than 3 million SNPs in the whole genome, and there are more than 3 million SNPs in the whole genome; in the rice genome, there is 1 SNP in about 300 bases. There are more than 1 million SNPs in the whole genome. [0003] SNPs have important application value in the research of human diseases and the guidance of animal and plant breeding. Utilizing the widely existing SNPs, an ultra-high-density genetic linkage map can be quickly constructed to achieve fine mapping of functional gen...

AI Technical Summary

Problems solved by technology

[0007] However, HRM technology also has some deficiencies that need to be improved

First of all, HRM technology relies on monitoring melting curves for DNA variation analysis, which has high resolution and sensitivity, but at the same time inevitably has the disadvantage of poor stability; secondly, HRM has high requirements for PCR amplification and requires the use of high-quality , DNA template with uniform concentration, and primers with good amplification specificity and efficiency; finally, because HRM analysis has high requirements on PCR products, the primers, amplification system and conditions used in PCR need to be continuously optimized

In summary, the preparation of high-quality template DNA and the need for repeated optimization of the PCR system are the main factors that limit the efficiency of HRM analysis

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