Mass acquisition method of different loci and flanking sequence thereof in genome DNAs (deoxyribonucleic acids)
A technology of differential sites and flanking sequences, which is applied in the field of obtaining differential sites and flanking sequences in genomic DNA in batches, can solve the problems of inability to widely apply genome differential analysis, high cost, and small analysis throughput, and achieves low cost, Simple and convenient operation, high efficiency
Active Publication Date: 2014-04-02
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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Problems solved by technology
Among them, although high-throughput sequencing technology and chip technology have a large analysis throughput, they are expensive and require special equipment and reagents, while the analysis throughput of other technologies is too small to be widely used in genomic differential analysis
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Embodiment 1
[0033] Example 1. Obtaining differential sites in genomic DNA in batches
[0034] 1. Obtaining genomic DNA
[0035] According to the method of phenol-chloroform extraction DNA in the literature (Lu Shengdong. Modern Molecular Biology Experimental Technology. Beijing: China Union Medical University Press, 1999), the coarse wool sheep (Altay sheep) and the fine wool sheep (Chinese Merino sheep) were extracted respectively. Genomic DNA from the ear tissue of Xinjiang Junken fine-wool sheep) with ddH 2 O The final concentration of genomic DNA was diluted to 200ng / μL.
[0036] 2. Hybridization of genomic DNA
[0037] Take the same amount of coarse and fine wool sheep genomic DNA obtained in step 1 (5 μL each, that is, 1 μg genomic DNA)) and mix them, and carry out molecular hybridization according to the following reaction system and conditions:
[0038] Hybridization reaction system: 1 μL 50×Denhardt’s (purchased from Invitrogen, catalog number 750018), 2.5 μL 20×SSC (solvent i...
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The invention discloses a mass acquisition method of different loci and flanking sequence thereof in genome DNAs (deoxyribonucleic acids). The method comprises the following steps: mixing a genome DNA A and a genome DNA B, hybridizing, digesting the single chain DNA area which has not form a double chain area by using CELI endonuclease, recovering the DNA segments within the target size range in the digestion production, and carrying out self-hybridization; and carrying out DNA terminal filling-in, linker DNA addition, PCR (polymerase chain reaction) amplification and sequencing to finally obtaining the different loci in the genome DNAs A and B. The method disclosed by the invention has the advantages of high efficiency, high flux and low cost, and one positive clone can provide information of 2 genome DNA different loci; and the method is simple and convenient to operate, does not need complex equipment or expensive reagents before sequencing, and can be widely used for detecting and analyzing genome DNA different loci among species and strains of animals, plants and microbes, and between cancer cells and normal cells.
Description
technical field [0001] The invention relates to a method for obtaining differential sites and flanking sequences in genome DNA in batches. Background technique [0002] In the process of species evolution, the genome differences between different species are mostly manifested in different genome sizes, chromosome rearrangements, gene structure changes, base insertions, base deletions, and a large number of single-base nucleotide mutations (SNP) ; Genome differences between different individuals of the same variety (line) are mainly manifested as insertions, deletions of several bases, and a large number of SNPs. These different forms of genome differences or mutations cause large phenotypic differences among different individuals, and mutations of some bases can even cause the occurrence of diseases and the termination of development of living organisms. The acquisition of genomic difference loci is of great significance for revealing the reasons for different phenotypes am...
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IPC IPC(8): C12N15/10C40B50/06
Inventor 甘尚权高蕊沈敏王新华刘守仁
Owner XINJIANG ACADEMY OF AGRI & RECLAMATION SCI