Reduced-representation genome library building method based on type IIB restriction enzyme digestion

A restriction endonuclease and genome technology, which is used in the field of simplified gene library construction based on type IIB restriction endonuclease digestion, can solve the problems of low library concentration, low linker connection efficiency, and uneven digestion.

Inactive Publication Date: 2015-04-29
SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the 2b-RAD methods recorded in the existing literature have disadvantages such as uneven digestion resulting in a small number of tags, low adapter ligation efficiency, and low library concentration.

Method used

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  • Reduced-representation genome library building method based on type IIB restriction enzyme digestion
  • Reduced-representation genome library building method based on type IIB restriction enzyme digestion
  • Reduced-representation genome library building method based on type IIB restriction enzyme digestion

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Experimental program
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Embodiment 1

[0037] 1. Design and synthesize the adapter and primer sequences in the following table for use in the library construction process of this example:

[0038]

[0039] 2. Genomic DNA extraction to be tested

[0040] Genomic NDAs from different individual melon leaves (respectively No. 1 melon genomic DNA, No. 2 melon genomic DNA, No. 3 melon genomic DNA, and No. 4 melon genomic DNA) were extracted as the objects for library construction in this embodiment. The extraction operation is briefly described as follows:

[0041] 2.1 Take 100 mg of fresh leaf tissue, add liquid nitrogen and grind it thoroughly.

[0042] 2.2 Extract using Tiangen Plant Genomic DNA Extraction Kit.

[0043] 2.3 Extracted DNA for quality inspection. Here it is detected by agarose gel electrophoresis, as attached figure 1 shown.

[0044] 3. Digestion

[0045] 3.1 Pre-experiment (take No. 1 melon genome DNA)

[0046] Take 7 copies of 500ng melon genomic DNA for enzyme digestion pre-experiment. The...

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Abstract

The invention relates to the technical field of molecular biology, and discloses a reduced-representation genome library building method based on type IIB restriction enzyme digestion. The method comprises steps as follows: (1), digestion is performed on genome DNA by means of BsaXI enzyme; (2), an adaptor I and an adaptor II are connected for an enzyme-digested product; (3), a connected product is purified; (4), PCR (polymerase chain reaction) amplification is performed on the purified product; (5), a PCR product is purified to obtain a sequencing library, and two adaptors of the step (2) are synthesized through hybridization of two nucleotide fragments represented by sequences SEQ ID NO: 1 and SEQ ID NO: 2 as well as two nucleotide fragments represented by sequences SEQ ID NO: 3 and SEQ ID NO: 2 respectively. The library building method is simple, fast and low in library building cost, the built library has high accuracy, and high-throughput sequencing can be more effectively performed.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a simplified gene library construction method based on type IIB restriction endonuclease digestion. Background technique [0002] Reduced genome sequencing is a high-throughput sequencing method for sequencing parts of the genome. Specifically, it refers to the use of bioinformatics methods to design a marker development plan, enrich fragments of specific length, and then apply high-throughput sequencing technology to obtain massive tag sequences to represent the entire genome information of the target species. [0003] The types of sequencing libraries commonly used in simplified genome sequencing can be summarized into three categories, ①simplified sequencing, including simplified libraries and simplified polymorphic sequence complexity; ②restriction site-associated DNA sequencing; ③low coverage genotyping library, Includes multiplex shotgun genotyping and sequencing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06
Inventor 孙子奎高文学丁方美王锋唐嘉婕
Owner SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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