Method for detecting single nucleotide polymorphism or small insertions and deletions by utilizing MutS proteins in genome range

A single nucleotide polymorphism, genomic technology, applied in the field of identifying and detecting mutation sites in DNA, can solve problems such as restricted application and inability to solve the problem of specific hybridization of genomic enzyme cut fragments

Inactive Publication Date: 2011-06-22
POMOLOGY RES INST GUANGDONG ACADEMY OF AGRI SCI
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Problems solved by technology

[0007] Most of the above methods are to study the detection of known or unknown mutation sites of single genes, because the

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Identification and detection of mutation sites.

[0040] 1. Extract the genomic DNA of Tianbao Gaojiao and Kangku No. 1 banana from banana leaves;

[0041] 2. Take the same amount of wild-type genomic DNA and mutant genomic DNA (5-10μg) as the research object;

[0042] 3. Using an appropriate amount of Taq I restriction endonuclease to completely digest the above-mentioned genomic DNA;

[0043] 4. Connect the connector, the connector sequence is:

[0044] SEQ ID NO:1 5'-CATGCTTGTAGACTCACA-3'

[0045] SEQ ID NO:2 3'-ACGAACATCTGAGTGTGC-5'

[0046] The lower chain of the adapter uses T4 polynucleotide kinase, and the ligation reaction time is 10 hours;

[0047] 5. Mixing: mix the two in equal amounts;

[0048] 6. Electrophoresis: electrophoresis on 2.0% agarose gel, the voltage is 20-30V, until the bromophenol blue indicator line reaches the front of the gel;

[0049] 7. Hybridization: Cut out the gel part containing DNA, place it in denaturing solution (0....

Embodiment 2

[0054] Example 2 Sequencing of mutation sites

[0055] 1. PCR amplification: Use the solution recovered by adsorption in Example 1 as a template to carry out PCR amplification. The primer sequence is SEQ ID NO: 3: CATGCTTGTAGACTCACA, the annealing temperature is 55°C, and the extension time is 1.0 minutes; the electrophoresis results are detected by electrophoresis , and recover the amplified fragments for sequencing.

[0056] Sequencing results: The amplification results were sequenced and compared, and a mutation was found in a NBS-LRR disease-resistant gene fragment. The sequence is as follows (the underlined bases represent the mutation sites):

[0057] Tianbao Gaojiao SEQ ID NO:4 gttactcgga gagttaattc ggtgcgcatc taacaaatct gaaggag G tg aagccaaaca

[0058] Kangku No. 1 SEQ ID NO:5 gttactcgga gagttaattc ggtgcgcatc taacaaatct gaaggag C tg aagccaaaca

[0059] Tianbao Gaojiao SEQ ID NO:6 agaatctttc gagggacaaa gcaaatcaga attggaaatc aaacttgcct ctctcctcac

[0060] Kangku No....

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Abstract

The invention belongs to the field of biotechnology, and relates to a method for detecting single nucleotide polymorphism or small insertions and deletions by utilizing MutS proteins in a genome range, which comprises the following steps of: mixing a DNA molecule to be detected and a wild DNA molecule, performing modification and renaturation, and realizing specific hybridization of correspondingsegments of the DNA molecule to be detected and the wild DNA molecule; adding biotin-labeled MutS proteins, and recovering the proteins to complete the identification and extraction of mutational sites; and performing polymerase chain reaction (PCR) amplification and sequencing on the extracted substances to detect and analyze mutant genes. Compared with the conventional method, the method is more simple, convenient and sensitive, can accurately identify and detect the mutational sites, and is high in reliability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for identifying and detecting mutation sites in DNA. Background technique [0002] Mismatch repair system MMR maintains the stability of genetic material. Mismatch recognition protein MutS is the first protein in the mismatch repair system to perform repair functions, and has the ability to recognize and bind mismatched base pairs. Due to the special function of MutS protein specific binding mismatch, the special function of specific binding mismatch makes it have great application potential in the research of detecting mutation and SNP. [0003] At present, the mismatch repair system of bacteria has been studied thoroughly. The mismatch repair system of E. coli contains more than ten kinds of proteins, namely MutS, MutL, MutH, DNA helicase II (DNA helicase II), single-strand binding protein exonuclease I (exonuclease I), exonuclease VII ( exonuclease VII ), exonuclease X (...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 李春雨孙清明易干军
Owner POMOLOGY RES INST GUANGDONG ACADEMY OF AGRI SCI
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