Method for improving dcaps marker detection efficiency

Abstract

The invention provides a method for improving dcaps marker detection efficiency, and relates to the technical field of molecular biology. The method for improving the dcaps marker detection efficiencycomprises the following steps: S1, selecting a target gene sequence: extracting a known wheat leaf rust resistance gene Lr67 and a wild type susceptibility gene thereof; S2, designing a dCAPS primer:extracting the wheat leaf rust resistance gene Lr67 in S1 to be matched with dCAPS Finder2.0 equipment to prepare a primer with a normal sequence length; and S3, amplifying the primer sequence length: amplifying the primer prepared in S2. According to the method for improving the dcaps marker detection efficiency, enzyme cutting sites are introduced into primers of dCAPS, and the length of the primers is generally about 20 bp, so that fragments which are about 20 bp shorter than original fragments are generated after PCR enzyme cutting, and polyacrylamide gel electrophoresis is generally adopted for separation so as to distinguish the difference of the fragments of 20 bp. According to the invention, a tail sequence of 30bp or above is added to the 5' end of each primer, so that the fragment size difference between a wild type and a mutant after enzyme digestion is increased to 50bp or above.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for improving the detection efficiency of dcaps markers. Background technique [0002] Single Nucleotide Polymorphisms (Single Nucleotide Polymorphisms, SNP) is the most widely distributed and the largest number of genetic markers, when the SNP site happens to be located at the restriction enzyme site, the polymorphic sequence can be amplified by enzyme digestion (Cleaved Amplified Polymorphic Sequences, CAPS) technology to detect, but this case is relatively rare, (Derived Cleaved Amplified Polymorphic Sequences, dCAPS) technology is an extension of CAPS, by introducing mismatched bases when designing primers near the SNP, thereby generating or removing restrictions Detection of endonuclease recognition sites, almost all SNP sites can be detected by CAPS and dCAPS technology, but in the prior art, the mismatched bases introduced by dCASP technology are in the ...

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Problems solved by technology

[0002] Single Nucleotide Polymorphisms (Single Nucleotide Polymorphisms, SNP) is the most widely distributed and the largest number of genetic markers, when the SNP site happens to be located at the restriction enzyme site, the polymorphic sequence can be amplified by enzyme digestion (Cleaved Amplified Polymorphic Sequences, CAPS) technology to detect, but this case is relatively rare, (Derived Cleaved Amplified Polymorphic Sequences, dCAPS) technology is an extension of CAPS, by introducing mismatched bases when designing primers near the SNP, thereby generating or removing restrictions Detection of endonuclease recognition sites, almost all SNP sites can be detected by CAPS and dCAPS technology, but in the prior art, the mismatched bases introduced by dCASP technology are in the primer sequence, that is, the enzyme cleavage site is in Near the end of the primer, the length of the general primer is about 20bp, so the size difference of the fragment size of the digested product is generally about 20bp, and it is very difficult to separate such a difference by agarose electrophoresis. , it takes more time and effort for complex gel preparation and staining, therefore, the detection efficiency of dCAPS is limited in practical applications, making the actual marker detection efficiency lower

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