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Sequencing library construction method for detecting lentivirus insertion sites, and lentivirus insertion site detection method

A technology of inserting sites and sequencing libraries, which is applied in the field of gene analysis and detection, can solve the problems of low binding efficiency of biotin affinity chromatography, increase of synthesis cost and working time, and high initial amount of template, so as to reduce synthesis cost and purify The effect of cost, low cost, and small initial amount

Pending Publication Date: 2021-06-29
深圳市禾沐基因生物技术有限责任公司
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  • Application Information

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Problems solved by technology

However, due to the low binding efficiency of biotin affinity chromatography and the relatively high initial amount of template required, it is difficult to apply to preclinical or clinical samples
And the biotin-labeled target region is used as a primer, which also increases the synthesis cost and working time

Method used

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  • Sequencing library construction method for detecting lentivirus insertion sites, and lentivirus insertion site detection method
  • Sequencing library construction method for detecting lentivirus insertion sites, and lentivirus insertion site detection method
  • Sequencing library construction method for detecting lentivirus insertion sites, and lentivirus insertion site detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] The insertion site analysis of the lentiviral vector in the genome of the 293T cell line comprises the following steps:

[0073] 1. Genomic DNA Extraction

[0074] 1.1 Collect the cells infected with lentivirus, wash them twice with PBS, add 0.05% trypsin to digest for 3 minutes, collect the cells and centrifuge, and discard the supernatant.

[0075] 1.2 Use Tiangen Blood Cell Tissue Genome Extraction Kit for genome extraction.

[0076] 1.3 Use Qubit to measure DNA concentration.

[0077] 2. Random interruption of DNA

[0078] 2.1 Take 100ng~1μg of DNA from the sample for fragmentation. If there is RNA contamination, add 1-2µl RNase for digestion before fragmentation, and quantify to 80-100µl for later use. Ultrasonic instrument CovarisLE210, Dutyfactor (%) 20, Cyelesperburst200, Waterlevel6, Intensity5, 105s.

[0079] 2.2 Use magnetic beads to purify the interrupted fragments, mix with the interrupted product according to the ratio of 0.6 / 0.2, let it stand at room ...

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Abstract

The invention relates to a sequencing library construction method for detecting lentivirus insertion sites, and a lentivirus insertion site detection method. The sequencing library construction method comprises the following steps: extracting genome DNA from lentivirus infected cells; carrying out fragmentation on the genome DNA and processing the genome DNA into a form suitable for linker connection; connecting asymmetric double-chain connectors to two ends of the fragmented genome DNA, wherein the asymmetric double-chain connectors comprise a long-chain sequence and a short-chain sequence; carrying out first-round PCR amplification on a joint connection product; performing second-round PCR amplification on a product of the first-round PCR amplification; and cyclizing the product of the second-round PCR amplification to obtain a cyclized sequencing library suitable for computer sequencing. The methods have the characteristics of simplicity in operation, short experiment time, low cost, small initial quantity, high flux, multiple analyzable aspects and the like, and can better perform accurate analysis and evaluation on the virus insertion site in gene therapy.

Description

technical field [0001] The invention relates to the technical field of gene analysis and detection, in particular to a method for constructing a sequencing library for detecting insertion sites of lentiviruses and a method for detecting insertion sites of lentiviruses. Background technique [0002] Gene therapy is an effective method for the treatment of genetic diseases and other malignant diseases, and has been clinically applied in the treatment of thalassemia major and blood diseases. Lentiviral vectors are commonly used transport vectors in gene therapy. However, due to the random integration of lentiviruses into the host genome, random insertion may inactivate tumor suppressor genes or activate the expression of proto-oncogenes, leading to the risk of carcinogenesis. In preclinical research and clinical application, the evaluation of lentiviral insertion sites and related safety is a necessary work and research content. It is very necessary and important to carefully ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6869
CPCC40B50/06C12Q1/6869C12Q2531/113C12Q2535/122
Inventor 李静孙海汐刘超周子恒杨林欧阳文杰刘田宾董国艺顾颖侯勇
Owner 深圳市禾沐基因生物技术有限责任公司
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