Method for detecting single nucleotide polymorphism or small insertions and deletions by utilizing MutS proteins in genome range
A single nucleotide polymorphism and genome technology, which is applied in the field of identification and detection of mutation sites in DNA, can solve the problems of limited application and inability to solve the specific hybridization of genome enzyme fragments
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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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