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Trace shellfish DNA extraction method for high-throughput sequencing library preparation

A technology for sequencing libraries and extraction methods, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of inability to guarantee the quality of high-throughput sequencing library preparation, affecting endonuclease activity, deformity damage, etc. Ensuring high-quality library preparation, reduced DNA extraction time, and easy and fast operation

Active Publication Date: 2019-10-18
OCEAN UNIV OF CHINA
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Problems solved by technology

[0005] The DNA extraction methods for marine molluscs are mainly divided into two types: one is represented by the traditional phenol-chloroform extraction method and kit, this method The complete and pure genomic DNA of the main band can be obtained, but a large amount of sample material is required, which may cause death or deformity of the shellfish, and the cost is high, so it is not suitable for molecular marker-assisted breeding and other studies that need to maintain good growth conditions of the research objects field; the other is a type of technology represented by the rapid extraction method of shellfish DNA, which can quickly obtain genomic DNA in a short time for ordinary PCR detection without affecting the survival of live scallops, but the DNA obtained by this method has different The degree of degradation, and contains more SDS, proteinase K and other enzyme inhibitors, which are easy to affect the activity of endonucleases, and cannot guarantee the preparation quality of high-throughput sequencing libraries, so the method of obtaining DNA by this method cannot be directly converted to preparation. In the application of high-throughput sequencing library

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  • Trace shellfish DNA extraction method for high-throughput sequencing library preparation
  • Trace shellfish DNA extraction method for high-throughput sequencing library preparation

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Embodiment 1

[0032] A method for extracting trace shellfish DNA for high-throughput sequencing library preparation, specifically comprising the following steps:

[0033] (1) Add 99.5 μl of cell lysate (100 mM NaCl, 10 mM Tris-HCl and 1 mM EDTA, pH=8.0) into a 1.5 ml EP tube, and then add 0.5 μl of proteinase K at a concentration of 20 mg / ml to the lysate , adjusted so that the final proteinase K enzyme concentration of the final mixture reached 0.1 μg / μl to prepare a mixture.

[0034] (2) Put 5.0 mg of gill filaments of isolated Ezo scallop into the EP tube.

[0035] (3) Put the above-mentioned EP tube into a 37-65°C water bath for 1.0h for digestion. This example is a 56°C water bath. In order to promote cell lysis and protein digestion, take out the EP tube and mix it upside down every 30 minutes.

[0036] (4) After digestion, absorb the supernatant and dilute it with triple steamed sterilized water at a ratio of 1:49. The diluent is the scallop genome DNA extracted by the present inve...

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Abstract

The invention discloses a trace shellfish DNA extraction method for high-throughput sequencing library preparation. Micro shellfish gill filament tissue is selected, only a cell lysis solution and protease K are needed for cell lysis, protein digestion and supernatant dilution, and obtained genome DNA can be directly used for preparing a high-throughput sequencing library. The tissue amount required by DNA extraction is as low as 5 mg, the operation is simple and rapid, and the method is particularly suitable for preparation of the high-throughput sequencing library of the micro tissue. The library prepared from the DNA sample is used for sequencing to obtain genotyping data, and the typing accuracy is up to 99.94%. The DNA extraction operation time is not longer than 30 minutes, and the extracted DNA is suitable for preparation of the high-throughput sequencing library of a large quantity of shellfish trace samples and subsequent whole genome typing analysis.

Description

technical field [0001] The invention relates to the technical field of deoxyribonucleic acid extraction and molecular marker typing, and more specifically relates to a method for extracting trace shellfish DNA for high-throughput sequencing library preparation. Background technique [0002] At present, shellfish accounts for nearly 80% of the total amount of marine aquaculture in my country, and is the pillar industry of marine aquaculture in my country. In recent years, with the rapid development of biological high-tech, how to efficiently use genomics and genetic informatics to create excellent germplasm of shellfish has become a frontier focus in the field of shellfish genetics and breeding. In addition, genome-wide SNP marker typing can provide abundant data information for the analysis of germplasm resources of precious shellfish resources such as giant clams and the development and protection of genetic resources. [0003] With the reduction of sequencing costs and th...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2527/125C12Q2535/122C12Q2521/301C12Q2563/185Y02A40/81
Inventor 王师张天琦刘平平吕佳马岑包振民
Owner OCEAN UNIV OF CHINA
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