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Single-cell Whole Genome Amplification and Library Construction Method for Improving Genome Coverage

A construction method and single-cell technology, applied in the field of molecular biology, can solve the problems of unsatisfactory single-cell whole-genome amplification methods, and achieve the effect of improving coverage

Active Publication Date: 2021-05-07
ZHEJIANG ANNOROAD BIO TECH CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in terms of genome coverage, the above three single-cell whole-genome amplification methods are not ideal, the highest is only about 60%, and there is a trend of MDA>MALBAC>DOP-PCR [4]

Method used

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  • Single-cell Whole Genome Amplification and Library Construction Method for Improving Genome Coverage
  • Single-cell Whole Genome Amplification and Library Construction Method for Improving Genome Coverage
  • Single-cell Whole Genome Amplification and Library Construction Method for Improving Genome Coverage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] (1) Single-cell gene amplification

[0061] A. Processing sorted cells:

[0062] Cells were sorted directly into 3 μL PBS in a clean bench without freeze-thawing.

[0063] B. Single-cell whole-genome amplification:

[0064] MDA was used for whole genome amplification, and Qiagen, REPLI-g Single Cell Kit was selected as the kit.

[0065] a) Prepare DLB buffer: Add 500 μL of water to the provided tube, mix thoroughly, and centrifuge briefly. DLBbuffer can be stored at -20°C for 6 months.

[0066] b) All buffers and reagents should be vortexed before use.

[0067] c) Adjust the temperature of the thermal lid of the PCR instrument to 70°C.

[0068] d) Prepare a sufficient amount of Buffer D2 (denatured buffer). Buffer D2 can be stored at -20°C for up to 3 months.

[0069] 3 μL of Buffer D2 is required for each reaction

[0070]

[0071] e) Make up the cell volume to 4 μL with PBS provided by the kit.

[0072] f) Add 3 μL Buffer D2, mix gently, and centrifuge brie...

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Abstract

The invention provides a single-cell whole-genome amplification and library construction method for improving genome coverage. The single-cell genome amplification method of the present invention adopts a multiple displacement amplification method to amplify the single-cell genome, which includes: contacting a set of primers, DNA polymerase and the single-cell genome in a solution; amplifying, so that the amplification reaction of the single-cell genome is carried out; wherein, the time of the amplifying is more than 0.5 hours and less than 2 hours.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a single-cell whole-genome amplification and second-generation sequencing library construction method capable of improving genome coverage. Background technique [0002] Single-cell sequencing technology (single-cell sequencing) was awarded the annual technology of "Nature Mathods" in 2013, and has broad application prospects in the fields of cancer, assisted reproduction and immunology. [0003] Single-cell genome sequencing is a technique for amplifying and sequencing the whole genome at the single-cell level. High-throughput sequencing for revealing cell population differences and cell evolution relationships. [0004] The genome of a single cell is extremely small. In order to meet the requirements of library quality control, on-machine sequencing and other detection, whole genome amplification must be performed to achieve linear or exponential growth of the geno...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C40B50/06C12Q1/6844
CPCC12Q1/6806C40B50/06
Inventor 梁峻彬张超洪燕刘涛玄兆伶李大为陈重建
Owner ZHEJIANG ANNOROAD BIO TECH CO LTD
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