Sequencing primer group and PCR-based whole genome sequencing method
A technology of whole genome sequencing and sequencing primers, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of slow speed, complicated operation and high requirements of SNP sites, and reduce the cost of sequencing , Simple library construction and sequencing, good enrichment effect
Active Publication Date: 2020-10-13
浙江物种链生物科技有限公司 +1
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Problems solved by technology
[0010] The purpose of the present invention is to provide a sequencing primer set and a PCR-based whole-genome sequencing method, aiming to solve the technical problems of slow speed, high requirements and complex operation of SNP sites in the existing chip screening genome
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Embodiment 1
[0070] A kind of whole-genome sequencing method based on PCR, is tested with the cassava of different varieties as example, and it comprises the steps:
[0071] Step 1: preparing the DNA of the sample to be tested;
[0072] Genomic DNA was extracted from different varieties of cassava that had consistent growth under the same growth conditions, the same growth period, the same part, and no pests and diseases. Long-term storage of samples requires liquid nitrogen or a refrigerator below -70°C. Genomic DNA was extracted using DNeasy 96 Plant Kit (QIAGEN) kit.
[0073] Quality detection and quantification of extracted genomic DNA: λmarker is used as a marker for agarose gel detection, 1 μL of DNA is taken, 2 μL of l0×bromophenol blue loading buffer is added, mixed well, and 0.8% goldview dye containing 0.5 μg / ml is injected In the agarose gel, use 1×TAE buffer solution, 90V electrophoresis for 40min; gel imaging analysis system (Tanon4100) to observe the DNA bands.
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The invention provides a sequencing primer group. The sequencing primer group comprises a general upstream primer, a general downstream primer and an enrichment promoter downstream primer. The invention also provides a PCR-based whole genome sequencing method. The method comprises the following steps: carrying out first PCR amplification on DNA of a sample to be detected by using the general upstream primer and the general downstream primer to obtain a first amplification product, carrying out second PCR amplification on the DNA of the sample to be detected by using the general upstream primerand the enrichment promoter downstream primer to obtain a second amplification product, and performing sequencing at the same time. According to the method, by optimizing the primers, the size of a genome and the size of the sample amount can be adjusted at will, and meanwhile the requirement for the DNA quality is not high; library building is simpler; a known SNP marker is not needed, and a chip does not need to be constructed in the earlier stage; the gene region enrichment effect is realized, and the whole genome detection and screening can be met.
Description
technical field [0001] The invention relates to the field of gene sequencing, in particular to a sequencing primer set and a PCR-based whole genome sequencing method. Background technique [0002] Genotyping is a series of genetic analyses, including molecular marker discovery and genotyping using high-throughput sequencing technology, one of the most powerful applications is in the field of plant breeding, opening up new possibilities for plant breeding and plant genetics research sex. It provides a cost-effective whole-genome scanning and multiplexed sequencing platform. Simplified sequencing technology is a new application of high-throughput sequencing to mine and genotype single nucleotide polymorphisms (SNPs) in species populations. Bioinformatics tools are needed to analyze and interpret reduced sequencing datasets. It is a low-cost technology and an excellent MAS tool. Simplified sequencing has been successfully applied to large-scale plant molecular breeding strat...
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IPC IPC(8): C12N15/11C12Q1/6869
CPCC12Q1/6869C12Q2531/113C12Q2535/122
Inventor 夏志强邹枚伶
Owner 浙江物种链生物科技有限公司