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Single-cell genome analysis method and kit

A genome analysis and single-cell technology, which is applied in the field of biological single-cell genome research, can solve the problem of less sequencing of single-cell genomes, and achieve the effect of simple operation and avoiding waste

Active Publication Date: 2012-07-04
BGI SHENZHEN CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies and reports on single-cell genome sequencing of mammals and other higher organisms

Method used

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  • Single-cell genome analysis method and kit
  • Single-cell genome analysis method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Single-cell genome studies in human cell lines

[0072] (1) Separation and lysis of single cells: add the immortalized human lymphocyte line single cells to the PBS droplet on the culture dish, dilute appropriately (that is, dilute according to the concentration of the cell line cells, until observed in the 200X field of view of the microscope until the number of cells is between 10-20), single cells were separated by oral suction under a microscope, and the obtained single cells were placed in a PCR tube containing 1.5-2uL ALB (Alkaline Lysis Buffer, specific formula: 50mMDTT, 200mM KOH), Place at -20°C to -80°C for at least 30min.

[0073] Heat the PCR tube containing single cells at 62°C-68°C, preferably 65°C, for 8-12min to lyse the cells and release the chromosomal DNA.

[0074] (2) Whole Genome Amplification (WGA):

[0075] Any of the following methods of multiple displacement amplification (MDA) or DOP-PCR whole-genome amplification can be used.

[0076] ①Multip...

Embodiment 2

[0103] Human Housekeeping gene primers for PCR detection of single cell WGA products

[0104] The single cells isolated from human tissue or blood are lysed and treated with WGA (refer to Example 1 for the specific treatment process), and the products are detected by Housekeeping Gene PCR.

[0105] The selected Housekeeping Gene and its corresponding primer information are as follows:

[0106]

[0107]

[0108] The aforementioned Housekeeping Gene is described in Eli Eisenberg and Erez Y. Levanon, (2003) Human housekeeping genes are compact. Trends in Genetics. 19(7): 362-365. The inventors found that, compared with other Housekeeping Genes on other chromosomes, the above selected Housekeeping Gene and its primers can be conveniently and accurately used for the qualitative detection of WGA amplification products in human cells.

[0109] The PCR system contains: heat-resistant DNA polymerase with 3'exolytic activity; single-cell WGA product (template); dNTP mix; Mg 2+ ;...

Embodiment 3

[0132] Genome Study of Pollen Cells of P. amabilis

[0133] This technique was applied to the genome research of P. amabilis pollen cells. It is intended that after the genome is amplified, the sample volume is sufficient to construct the Solexa DNA library, and finally sequenced on the machine.

[0134] Considering that there are 3 to 5 single cells in the pollen grains of plants, first separate the pollen grains one by one under an inverted microscope, add a mixed solution of 4% cellulase and 2% pectinase equal to the volume of the pollen grains, and place Mix for 6 hours at room temperature in the dark on a vertical mixer to lyse and break the cell wall. After completion, add the lysis system of the same pollen grain to the PBS droplet on the petri dish, separate single cells under the microscope, and put the obtained single cells into a PCR tube containing 1.5uL ALB (alkaline lysis buffer).

[0135] Subsequently, the MDA (multiple displacement amplification) reaction was...

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Abstract

The invention discloses a single-cell genome analysis method and a kit. The method comprises the following steps of: a, acquiring a complete cell genome DNA (Deoxyribonucleic Acid); b, carrying out single-cell whole genome amplification; c, carrying out quantitative detection and qualitative detection, wherein the qualitative determination comprises that single-cell whole genome amplification product is determinated by adopting a Housekeeping Gene detection method. The kit contains a specific primer of the single cell Housekeeping Gene. By adopting the method disclosed by the invention, genetic variation information of a single cell genome can be comprehensively and completely analyzed, and an effective research strategy can be provided for the single cell genome of a brand-new specie; meanwhile, qualitative and quantitative detection screening steps are introduced, and many unqualified amplification samples can be removed, thus quality of a downstream upper computer sequencing library is controlled and unnecessary waste can be greatly avoided.

Description

technical field [0001] The invention relates to molecular cell biology, in particular to a technique and a kit capable of efficiently conducting research on single-cell genomes, especially single-cell genomes of higher organisms. Background technique [0002] Heterogeneity exists between different tissues of an individual and between different parts of the same tissue. There is also heterogeneity between cells, even in vitro cultured cell populations with the same genetic background. [0003] In order to better study cell biology, it is very necessary to develop technical methods applied to the study of single cells to reveal the rules of cell heterogeneity. Therefore, some scholars put forward the concept of "single-cell analysis (SCA)" and elaborated it from the perspective of "omics (Omics)". In single-cell analysis, the first thing to study is the single-cell genome. [0004] At present, trace DNA and single-cell genome research have been widely used in archaeology, m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q1/68C12Q1/6851C12Q1/6876C12Q2600/166C12Q1/6806C12Q1/6869C12Q2531/119C12Q2545/101
Inventor 殷旭阳鲍莉徐讯吴汉杰刘晓羽张秀清杨焕明
Owner BGI SHENZHEN CO LTD
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