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Preparation method of gene DNA (Deoxyribose Nucleic Acid) sequence capture probe

A technology of DNA sequence and capture probe, which is applied in the field of molecular biology, can solve the problems of high cost in the diagnostic market and the inability of whole-genome sequencing to enter the molecular diagnostic market, and achieve rapid and efficient preparation, concise operation steps, and high efficiency. Effect

Active Publication Date: 2014-07-02
HANGZHOU D A GENETIC ENG
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Problems solved by technology

[0005] The molecular diagnostic market is huge, but the diagnostic market has more stringent cost requirements. At least whole genome sequencing cannot enter the molecular diagnostic market for a long time. This is the opportunity for target sequence capture sequencing

Method used

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  • Preparation method of gene DNA (Deoxyribose Nucleic Acid) sequence capture probe
  • Preparation method of gene DNA (Deoxyribose Nucleic Acid) sequence capture probe
  • Preparation method of gene DNA (Deoxyribose Nucleic Acid) sequence capture probe

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Embodiment 1

[0062] Example 1: In order to test the effectiveness of the probe preparation method described in the present invention, the present invention selected a target gene GSTP1 (genbank number in the NCBI database is NM_000852.3), and tested the preparation of the capture probe for this gene. Effectiveness and efficiency, specifically, include the following steps:

[0063] 1. Design primers for the DNA sequence of the coding region of the target gene GSTP1 for PCR amplification:

[0064] Synthetic primers:

[0065] Seq1F: 5'-CTACACCGTGGTCTATTTCC-3'

[0066] Seq1R: 5'-CAGGGTGAGGTCTCCGTC-3'

[0067] Seq2F: 5'-CAAGTTCCAGGACGGAGA-3'

[0068] Seq2R: 5'-CGCCTCATAGTTGGTGTAGAT-3'

[0069] Seq3F: 5'-CATCTCCCTCATCTACACCAA-3'

[0070] Seq3R: 5'-CTCATGGATCAGCAGCAAGT-3'

[0071] Seq4F: 5'-CTTCGCTGACTACAACCTG-3'

[0072] Seq4R: 5'-ACTGTTTCCCGTTGCCATT-3';

[0073] The target fragment size is 180bp, and the amplification products are named A1-1, A1-2, A1-3, A1-4. For the amplification resu...

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Abstract

The invention discloses a preparation method of a gene DNA (Deoxyribose Nucleic Acid) sequence capture probe. According to the technical scheme, the method comprises the following steps: 1) designing two partially complementary primers and carrying out annealing treatment so as to form a partially double-chain structured split joint, and carrying out a coupled reaction between the split joint and an ordinary PCR (Polymerase Chain Reaction) amplification product (A1) of a target gene so as to obtain a target gene (A2) two ends of which are provided with joints; 2) designing corresponding primers according to the sequences of the joints and amplifying the target gene (A2) so as to obtain an amplification product (A3) with a T7 promoter; and 3) quantifying the product (A3), and adding NTP (Nucleotide Triphosphate) with biotin labels (wherein biotins are labeled on the NTP is labeled with biotins but ATP (Adenosine Triphosphate), CTP (Cytidine Triphosphate) and DTP (Deoxyadenosine Triphosphate) are not labeled with biotins) into the product (A3) under a certain condition so as to carry out an in-vitro transcription reaction, thereby finally obtaining an RNA (Ribose Nucleic Acid) probe with biotin labels (gene DNA sequence capture probe).

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for making probes that can be used for capturing multiple target gene sequences and performing next-generation sequencing. Background technique [0002] The development of next-generation sequencing technology has opened up a new situation for modern genomics research, making it possible for ordinary laboratories to sequence the entire human genome. However, the experimental cost of whole genome sequencing and the requirements for the analytical capabilities of researchers are still very high Because the functions of most genes are still not very clear, it will be a great challenge to conduct bioinformatics analysis on the massive data generated. [0003] Before the next-generation sequencing technology, the research on specific regions of large genome fragments was mainly carried out by traditional capillary sequencing after PCR walking. The problem with th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12Q1/6811C12Q2563/131
Inventor 谭海芹洪旭涛余文菁张珊珊余科宓娅娜
Owner HANGZHOU D A GENETIC ENG
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