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A preparation method of modified dna hybridization probe for targeted hybridization capture

A technology of hybridization capture and hybridization probe, which is applied in the field of genetic engineering, can solve the problems of RNA hybridization capture probes not having stability, etc., achieve the effect of wide application range and lower production cost

Active Publication Date: 2019-03-08
HANGZHOU LC BIOTECH
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Problems solved by technology

Among them, the oligonucleotide pool is synthesized on the microarray chip, and then the RNA probe is further prepared in vitro. Although the probe preparation is extremely flexible, the RNA hybridization capture probe is not stable and requires strict storage. and shipping conditions

Method used

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  • A preparation method of modified dna hybridization probe for targeted hybridization capture
  • A preparation method of modified dna hybridization probe for targeted hybridization capture
  • A preparation method of modified dna hybridization probe for targeted hybridization capture

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Embodiment approach

[0021] A method for preparing a modified DNA hybridization probe for targeted hybridization capture, comprising the following steps:

[0022] (1) Obtain DNA templates containing target sequences and universal primer regions by preparing oligonucleotide pools in parallel.

[0023] (2) Use Taq polymerase and PCR primers containing dUTP to carry out PCR reaction, and introduce biotin-labeled ATP and methylated dm during PCR 5 CTP; the PCR primer is a pair of primers, including a forward primer and a reverse primer, the sequence of the forward primer is 5'-GAGCTTCGGTTCACGCAATG-3' (SEQ ID No.1), and the sequence of the reverse primer is 5'-UGCCUAGGACCGGAUCAAC -3' (SEQ ID No.2), forward primer and reverse primer were synthesized by Shanghai Sangong. In the PCR reaction system, the final concentration of dUTP-containing PCR primers was 0.5 μM.

[0024] (3) The PCR product was purified by magnetic bead enrichment method.

[0025] (4) The purified PCR product was cleaved with USER a...

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Abstract

The invention discloses a preparation method of a modified DNA (deoxyribonucleic acid) hybridization probe for targeted hybrid capture. The preparation method mainly includes preparing a DNA template prior to PCR (polymerase chain reaction) amplification and purifying a PCR product; subjecting the purified PCR product to USER enzyme digestion and T4 DNA polymerase terminal blunting, wherein one of double-stranded DNA of an Lambda exonuclease hydrolysis product has 5'-phosphorylated single strand, so that bioti-labeled single-stranded DNA is obtained; purifying the bioti-labeled single-stranded DNA by a magnetic beads enrichment method to obtain the modified DNA hybridization probe. The preparation method of the modified DNA hybridization probe for targeted hybrid capture has the advantages that production cost is reduced greatly, and the preparation method is wide in application range including exon regions, intron regions, mitochondrion regions and the like of genomes of any species. The obtained DNA hybridization probe can be applied to construction and sequencing of next generation targeting libraries.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for preparing a modified DNA hybridization probe for targeted hybridization capture. Background technique [0002] In 2003, the "Human Genome Project" obtained the first human genome map, which led to the development of genomics analysis and personalized medicine. Along with the innovation of human technology, Next Generation Sequencing technology (Next Generation Sequencing) has appeared in people's field of vision, which can complete the sequence determination of hundreds of thousands to millions of DNA molecules at one time, making the genome detailed in a very short time. Research becomes possible. A large number of studies have shown that the occurrence of human diseases, especially cancer, has a significant correlation with the single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) of individual related genes. Although the cost of next-g...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12Q1/6806C12Q2525/117C12Q2563/143C12Q2563/149C12Q2565/519
Inventor 郎秋蕾周小川方超殷楠楠孟佳军
Owner HANGZHOU LC BIOTECH
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