Method and device for gene sequencing of plurality of mixed DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequences

A DNA sequence and DNA sequencing technology, applied in biochemical cleaning devices, biochemical equipment and methods, enzymology/microbiology devices, etc. Effect

Inactive Publication Date: 2013-04-17
SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA +1
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Problems solved by technology

However, for studying the diversity of TCR and BCR, the error rate of the existing sequencing technology is too high, so that i

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  • Method and device for gene sequencing of plurality of mixed DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequences
  • Method and device for gene sequencing of plurality of mixed DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequences
  • Method and device for gene sequencing of plurality of mixed DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequences

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Embodiment Construction

[0026] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

[0027] refer to figure 1 , figure 1 It is a flow chart of two embodiments of the method for performing gene sequencing on multiple mixed DNA sequences in the present invention, including:

[0028] Step S101: Mix M pairs of first upstream primer sequences with the first downstream primer sequences and a solution of N DNA sequences of TCR and / or BCR in the immune cell population, and perform two cycles of PCR reaction to obtain the first PCR product , wherein M and N are natural numbers, M is greater than or equal to one hundred times N, and the first upstream primer sequence or the first downstream primer sequence comprises a linker sequence, a tag sequence and a first upstream base sequence from the 5' end to the 3' end The primer sequence or the first downstream basic primer sequence, the label sequence of each pair of the first upstream primer seq...

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Abstract

The invention discloses a method and a device for gene sequencing of a plurality of mixed DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) sequences, which is mainly characterized by adding random labels at two ends of each DNA or RNA template sequence before establishing a bank. The method comprises the following steps of: mixing a first forward primer containing the random label and a connector, a first reverse primer and the DNA sequences, performing multiple PCR (Polymerase Chain Reaction) amplification on two PCR circulations, and purifying the PCR product to obtain a first DNA product; mixing the first DNA product, a second forward primer containing a sample indexing sequence and the connector, and a second reverse primer, performing PCR reaction to obtain a second PCR product; purifying to obtain a second DNA product; and sequencing the second DNA product to obtain a sequencing result of each DNA sequence. According to the method disclosed by the invention, by introducing the random labels to each DNA molecule, the sequencing precision is improved, the error rate of the sequencing is obviously reduced, and the copy number of each DNA is precisely detected.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and device for gene sequencing of multiple mixed DNA or RNA sequences. Background technique [0002] In the immune cell population, the diversity of T lymphocyte receptor molecules (TCR, T cell receptor) and B lymphocyte receptor molecules (BCR, B cell receptor) is hundreds of thousands to millions. The α and β chains that make up TCR or BCR not only have different structures, but the formation of each chain also undergoes the combination of many gene segments in different regions of V, J, or V, J, and D. [0003] When studying the diversity of TCR and BCR, it is necessary to sequence many gene fragments in the V, J or V, J, D regions of TCR and BCR. In general, existing sequencing technologies can be adapted to general gene sequencing requirements. However, for studying the diversity of TCR and BCR, the error rate of the existing sequencing technology is too hi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12M1/34
Inventor 李周芳贺建奎施国宁
Owner SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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