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Magnetic bead enrichment technique based method for high-flux development of genome SSR markers

A genomic, high-throughput technology, applied in biochemical equipment and methods, microbial assay/inspection, etc., which can solve problems such as low efficiency, high cost, and cumbersome process

Active Publication Date: 2014-12-17
SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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Problems solved by technology

[0008] Therefore, the technical problem to be solved in the present invention is to provide a high-throughput method for developing genomic SSR markers based on the magnetic bead enrichment method in view of the shortcomings of the current methods for developing SSR markers, which are cumbersome, costly, and inefficient.

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  • Magnetic bead enrichment technique based method for high-flux development of genome SSR markers
  • Magnetic bead enrichment technique based method for high-flux development of genome SSR markers
  • Magnetic bead enrichment technique based method for high-flux development of genome SSR markers

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Embodiment 1

[0056] Example 1 A method for high-throughput development of SSR markers in yellow cover flounder based on magnetic bead enrichment method

[0057] 1. Experimental materials: 4 liver tissue samples of Limanda aspera. Three yellow-covered flounder were killed, and the liver tissues were taken out, stored in anhydrous ethanol solution, and stored at room temperature. Among them, one mixed sample is the same amount of liver tissue mixture of three yellow Gai flounder individuals, and there is no reference genome sequence for this species.

[0058] 2. Genomic DNA extraction: Genomic DNA was extracted using the TIANamp Genomic DNA Kit kit, and the specific extraction method was referred to the kit’s instruction manual.

[0059] 3. Genomic DNA random fragmentation: the specific method includes the following steps:

[0060] (1) Fragmentation of genomic DNA using high-pressure nitrogen: Take 4 μg of genomic DNA, add TE buffer to 100 μl, add to the fragmentation cup, and then add 500...

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Abstract

The invention discloses a magnetic bead enrichment technique based method for high-flux development of genome SSR (simple sequence repeat) markers. The method comprises the steps of: (1) extracting target genome DNA; (2) subjecting the obtained genome DNA to random fragmentation; (3) constructing a genome random fragmented DNA library; (4) amplifying the DNA library; (5) utilizing a biotin labeled SSR probe to perform hybridization with a target fragment in the library; (6) carrying out streptavidin magnetic bead enrichment on the SSR sequence-containing DNA library fragments; (7) amplifying and purifying the enriched DNA library fragments; (8) subjecting the purified DNA fragments to emulsion PCR amplification and sequencing; and (9) searching sequences containing SSR loci in the obtained sequences. The method reduces the library construction difficulty, shortens the experimental period, improves the sequencing flux, lowers the sequencing cost, and can be widely used as an effective method for high-flux development of various species SSR markers.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for high-throughput development of genomic SSR markers based on a magnetic bead enrichment method. Background technique [0002] SSR (Simple Sequence Repeat), also known as Microsatellite DNA (Microsatellite DNA). The repeat unit is very short, only 1-6bp; the repeat number is variable, with a total length of tens of bp, distributed in different positions throughout the genome. The sequences at both ends of the microsatellite DNA are generally relatively conservative single-copy sequences, based on which specific primers can be designed for PCR amplification. Depending on the number of tandem repeats, polymorphisms in the length of microsatellite DNA can be revealed, and fragments with length polymorphisms can be used as molecular markers. Microsatellite markers are widely used in research fields such as genetic map construction, population genetics, fingerprint analysis, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2535/122C12Q2523/308
Inventor 孙子奎陈永灿
Owner SHANGHAI PASSION BIOTECHNOLOGY CO LTD
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