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Method for separating short interspersed repeated segments based on magnetic bead probe complexes

A repetitive sequence and compound technology, applied in recombinant DNA technology, DNA preparation, etc., can solve the problems of heavy workload, high cost, and low efficiency, and achieve the effects of reducing cost consumption, easy control, and improving experimental efficiency

Inactive Publication Date: 2009-03-11
INST OF AQUATIC LIFE ACAD SINICA
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  • Claims
  • Application Information

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Problems solved by technology

This traditional method requires a lot of work, takes a long time, is costly, difficult to operate, and low in efficiency

Method used

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  • Method for separating short interspersed repeated segments based on magnetic bead probe complexes
  • Method for separating short interspersed repeated segments based on magnetic bead probe complexes
  • Method for separating short interspersed repeated segments based on magnetic bead probe complexes

Examples

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Embodiment 1

[0024] Example 1: The whole process is illustrated in the case of bighead carp.

[0025] A. According to figure 1 It can be seen that the preparation of the genome fragment enrichment library. Step 1: Enzyme digestion of genomic DNA. About 40 micrograms of genomic DNA and 20 units of restriction endonuclease HaeIII were added to 100 microliters of enzyme digestion system, and digested overnight for complete digestion. The map after digestion is as figure 2A, Genomic DNA was uniformly digested below 4KB, and the recovered 500bp-2kb fragment was dissolved in 40 microliters of sterile water. The second step: add joints. The two oligonucleotides that generate the linker are A: 5P'GGCAGGATCCACTGAATTCGC-3' and B: 5'-AGCGAATTCAGTGGATCCTGCC-3'. The two oligonucleotides were carried out in sterile water with a final concentration of 10 μM to perform the following procedures to achieve the polymerization of the two strands to form a linker: 95°C for 3 minutes, 65°C for 2 minutes, ...

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Abstract

The invention discloses a method for separation, dispersion and sequence repetition of a bead-probe complex. The method comprises the following steps: firstly, the preparation of a genome fragment enriched library, which is to perform restriction enzyme digestion on a genome, to recover fragments with adequate dimensions, to add linkers on T4 ligase, and to perform cyclic amplification of the genome fragment library after restriction enzyme digestion through PCR of a small number of linkers; secondly, the preparation of the bead-probe complex, which is to realize biotin labeled DNA probes by the primer amplification method, and to bind biotin labeled single-chain probes on beads; and thirdly, acquisition of a target fragment group, which is to perform crossover on the bead-probe complex and the genome library, to acquire a target fragment, to separate and clone the target fragment, and to perform positive selection on the target fragment. The method is simple, high-efficiency and quick, shortens the experimental period, reduces the cost consumption, greatly improves the efficiency, and does not require special experimental equipment.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a method for rapidly and large-scale isolating short interspersed repeat sequences (SINE) from genomes. This method is based on the direct forward sorting of the magnetic bead sorting system. The probes are bound to the magnetic beads, and hybridized with the amplified enzyme-cut genome fragment library in solution to directly capture a large number of target fragments containing short interspersed repeat sequences (SINEs). . Background technique [0002] Short Interspersed Repeats (SINE) is a retrotransposon with a length of 50-500bp that widely exists in eukaryotic genomes. Its copy number is generally 10 4 -10 6 Left and right, scattered or clustered anywhere in the genome. A typical SINE consists of three parts: tRNA-associated region, family-specific region and tail region. SINE is considered to be an excellent marker for studying the phylogenetic relationship o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 童超波何舜平
Owner INST OF AQUATIC LIFE ACAD SINICA
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