Single cell whole genome amplification method

A whole-genome amplification and single-cell technology, which is applied in the field of single-cell genome analysis, can solve the problems of low amplification product, deviation and inhomogeneity of result analysis

Inactive Publication Date: 2016-02-03
SUZHOU BASECARE MEDICAL DEVICE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at the same time, this technology also has some limitations. Since the initial reaction product of whole genome amplification is very small, even only one DNA molecule, it is inevitable that there will be inhomogeneity in the amplification reaction, that is, there may be some in the genome. Loci are amplified multiple times, while others are not amplified, which may skew the results analysis
Therefore, many scientific research institutions are also committed to the continuous improvement of whole genome amplification technology. Some existing amplification technologies are mainly based on multiplex PCR and isothermal strand replacement technology, such as DOP-PCR, MDA, etc., but there are still amplification biases. Problems with large, low amplification products

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Isolation of Single Cell Samples

[0038] The cultured positive cell lines with abnormal chromosomes and normal cell lines were used to select individual cells to verify the experimental process. The cell types are shown in Table 3. Micromanipulation was used to separate individual cells, and a single cell was sucked in with a mouth pipette. Wash 3 times in 1×PBS solution, and then transfer to a 0.2mL PCR tube with a volume of no more than 2μl.

[0039] table 3

[0040] cell number

Embodiment 2

[0042] Single Cell Expansion Experiments

[0043] According to the single-cell amplification process described in the summary of the invention, the isolated single-cell sample was subjected to whole-genome amplification; the amplified product was purified with 1.5× AmpureXP magnetic beads; after purification, the concentration of the amplified product was detected with Qubit, and the result As shown in Table 3 in Example 1, the concentration of the amplified product is uniform and the repeatability is good; the length of the amplified fragment is analyzed by 2% agarose gel electrophoresis, and the results are as follows figure 1 shown.

Embodiment 3

[0045] Amplified product fragmentation

[0046] The purified product was fragmented with NEB's fragmentation enzyme digestion kit. The fragmentation length was 150-200bp, and treated at 37°C for 20 minutes. The enzyme digestion system is shown in Table 4:

[0047] Table 4

[0048] components

[0049] The digested product was purified with 1.8× AmpureXP magnetic beads.

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Abstract

The invention discloses a single cell whole genome amplification method, comprising following steps: (1), performing separating to obtain single cell samples; (2), subjecting the single cell samples to whole genome DNA amplification; (3), subjecting an amplification product to quantitative and qualitative analysis; (4), establishing a genome sequencing library, performing sequencing, and performing genome bioinformatic analysis. The single cell genome DNA amplification method is built and can be used to detect the single, tertiary, multiple and sex chromosome deficiency of a whole chromosome, the insertion and deficiency of few bases, DNA copy number variation, and the genetic variation information of single cell genomes such as single nucleotide polymorphisms.

Description

technical field [0001] The invention relates to a single-cell genome analysis method based on non-exponential amplification and high-throughput sequencing, through which the genome is analyzed at the single-cell level. Background technique [0002] With the continuous development of genomics research, more and more researchers are no longer limited to genetic research at the multi-cellular level, but are focusing on the more specific level of a single cell. Single cells are the basic unit of multicellular organisms. The development of multicellular organisms begins with a single cell and continues to divide in strict temporal and spatial sequences. However, such complex processes can only be truly understood at the single-cell level. In addition, each cell in a multicellular organism is in a unique microenvironment, and no two cells are exactly the same. Especially in tumor tissues, the existence of high heterogeneity makes it necessary to study from the perspective of singl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 梁波孔令印冒燕申静静刘慧敏孙丹凤
Owner SUZHOU BASECARE MEDICAL DEVICE CO LTD
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