Construction method of single-tube and high-flux sequencing library

A technology of sequencing library and construction method, which is applied in the field of construction of single-tube high-throughput sequencing library, can solve the problems of long time for library construction and low hybridization efficiency, and achieves the effect of low cost

Active Publication Date: 2016-02-17
XIAMEN SPACEGEN BIOTECH CO LTD
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Problems solved by technology

The disadvantage of this method is that the hybridization efficiency is low, and the library construction takes about 24 hours.

Method used

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  • Construction method of single-tube and high-flux sequencing library
  • Construction method of single-tube and high-flux sequencing library
  • Construction method of single-tube and high-flux sequencing library

Examples

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Embodiment 1

[0040] In this example, the method of the present invention is used to design the library construction system for the common mutation sites of 5 genes related to the current tumor field. At the same time, the high-throughput sequencer IontorrentPGM platform is used to detect EGFR, KRAS, BRAF, PIK3CA, HER2, TP53 and other gene mutations. .

[0041] material:

[0042]

[0043]

[0044] 1. Preparation of samples and reference substances

[0045] Receive clinical paraffin tissue samples, cut the samples into 5-10μm, add 1ml of xylene to dewax, centrifuge to collect the precipitate, add 1ml of absolute ethanol to the precipitate, dry at room temperature or 37℃, add proteinase K and BufferATL, 56℃ Digest and lyse for 1 hour, incubate at 90℃ for 1 hour, add 200ml BufferAL and mix well, then add 200μL of absolute ethanol to mix well, carefully transfer the supernatant to QIA2ml spin column, leave at 6000×g (8000rpm) for 1min, add 500μL BufferAW1, 6000×g (8000rpm) for 1min, carefully open t...

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Abstract

The invention discloses a construction method of a single-tube and high-flux sequencing library. The method comprises the following steps: 1, respectively designing multiple pairs of basic amplimers for multiple target genes; 2, designing a pair of asymmetrically connected probes and a general primer not complementary to a mankind genome; 3, amplifying a template, the multiple pairs of basic amplimers, the asymmetric probes and the general primer in a PCR reaction system containing a DNA ligase and a DNA end modification enzyme to obtain an amplification product, wherein the amplification program sequentially comprises initial denaturation, first stage amplification and second stage amplification; and 4, purifying the amplification product to obtain the high-flux sequencing library. The construction method of the single-tube and high-flux sequencing library realizes single-tube rapid library completion of multiple target sequences can effectively solve the difficulty of polygene and multi-target detection of somatic cells of present clinic tumors, genetic diseases and other diseases on the basis of a small amount of clinic samples through combining a high-flux sequencing platform, and has low cost.

Description

Technical field [0001] The invention belongs to the technical field of detection biology, and specifically relates to a method for constructing a single-tube high-throughput sequencing library. Background technique [0002] Molecular biology diagnostic technology is the crystallization of the tremendous progress made in modern molecular biology and molecular genetics. It is produced on the basis of people's deepening understanding of the structure of genes and the essential issues of life such as gene expression and regulation. In recent years, the methodological research of molecular biology diagnostic technology has made great progress, and methods such as restriction endonuclease zymography analysis, nucleic acid molecular hybridization, and restriction fragment length polymorphism linkage analysis have been established successively. In 1985, the DNA in vitro amplification technology (PolymeraseChain Reaction, PCR), which was founded by Mullis and others in the Human Genetics ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 陈琰郭飞飞
Owner XIAMEN SPACEGEN BIOTECH CO LTD
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