Kit for single cell genome-wide amplification and application thereof

A single cell and kit technology, which is applied in recombinant DNA technology, microbial measurement/testing, DNA preparation, etc., and can solve the problems of high amplification costs and other issues

Inactive Publication Date: 2016-04-13
MGI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the accompanying high expansion cost has also be...

Method used

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  • Kit for single cell genome-wide amplification and application thereof
  • Kit for single cell genome-wide amplification and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, the preparation of kit

[0057] The kit consists of cell denaturation lysate, neutralization buffer and amplification system.

[0058] Cell denaturation lysate: composed of solute and solvent; the solvent is water; the solute and its concentration are as follows: 0.1mMKOH, 1mMEDTA and 0.1MDTT.

[0059] Neutralization buffer: composed of solute and solvent; solvent is water; solute and its concentration are as follows: 60mMKH 2 PO 4 and 5mMK 2 HPO 4 .

[0060] Amplification system (40.3μl): from 30μl H 2 O, 5 μl 10×Phi29buffer, 2 μl ldNTP solution, 2 μl N8primers solution, 0.25 μl 20 mg / ml BSA aqueous solution, 0.05 μl 10% PluronicF68 solution and 1 μl Phi29 DNA polymerase solution.

[0061] 10×Phi29buffer: CompleteGenomics, Cat. No. MP00012. dNTP solution (in the dNTP solution, the concentration of dATP / dTTP / dCTP / dGTP is 10 mM): FERMENTAS, Cat. No. R0193. N8primers solution (N8primers are random primers, composed of 8 deoxyribonucleotides, AGCT ran...

Embodiment 2

[0062] Embodiment 2, the application of kit

[0063] 1. Sampling

[0064] Peripheral blood was collected from a normal female with informed consent. On the one hand, the genomic DNA (named as control genome) of peripheral blood is extracted. On the other hand, density gradient centrifugation is used to separate lymphocytes, and then single lymphocytes are separated by mouth pipette.

[0065] 2. Preparation of single-cell genome amplification products using the kit prepared in Example 1

[0066] 1. Get the single lymphocyte obtained in step 1, add 3.7 μl of PBS buffer (pH7.5, 0.1M PBS buffer) and 3 μl of the cell denaturation lysate in the kit prepared in Example 1 (the purpose is to lyse the cells and Release the genomic DNA therein), let stand at 65°C for 10 minutes, then add 3 μl of the neutralization buffer in the kit prepared in Example 1 (the purpose is to terminate cell lysis and return the system to a neutral environment), 9.7 µl of template solution was obtained. ...

Embodiment 3

[0086] Embodiment 3, the application of kit

[0087] 1. With the informed consent of the pregnant woman, collect the umbilical cord blood of an abnormal fetal sample (both non-invasive prenatal testing and amniocentesis showed 47,+18,XY), and use density gradient centrifugation to separate mononuclear cells, and then apply Flow cytometry separates individual lymphocytes therein.

[0088] 2. Get the single lymphocyte obtained in step 1, add 3.7 μl of PBS buffer (pH7.5, 0.1M PBS buffer) and 3 μl of the cell denaturation lysate in the kit prepared in Example 1 (the purpose is to lyse the cells and Release the genomic DNA therein), let stand at 65°C for 10 minutes, then add 3 μl of the neutralization buffer in the kit prepared in Example 1 (the purpose is to terminate cell lysis and return the system to a neutral environment), 9.7 µl of template solution was obtained.

[0089] 3. After completing step 2, take 9.7 μl of the template solution (the DNA content is about 6 ng), add i...

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Abstract

The invention discloses a kit for single cell genome-wide amplification and application thereof. The invention protects a kit for single cell genome amplification first, comprising a second amplification system; the second amplification system is composed of 10*Phi29buffer, water and a first amplification system; the first single amplification system is composed of dNTP (1.8-2.2)*10-8 mol, N8 primers (1.8-2.2)*10-11 mol in total concentration, BSA (4.5-5.5)*10-3 mg, Pluronic F68 0.0045-0.0055Mul, and DNA polymerase 9-11 U Phi29; the N8 primers are random primers, composed of eight deoxyribonucleotides, in a random arrangement of A, G, C and T. The WGA kit low in cost, high in efficiency and stable in amplification effect is developed based on the multiple exchange amplification principle and amplification features of DNA polymerase Phi29, and the kit has an important applicable prospect and polarization value.

Description

technical field [0001] The invention relates to a kit for single cell whole genome amplification and application thereof. Background technique [0002] With the development of flow cytometry and laser microdissection technology, it is relatively easy to isolate single target cells. Single cells have all the genome information of the species. Compared with genome samples obtained from multiple cells, genome samples obtained from single cells have no defects such as cell heterogeneity, so single cells have important genetic analysis application value. With the maturity and popularization of the second-generation high-throughput sequencing technology, genetic analysis based on gene sequencing has also developed. However, the most basic requirement of most genetic analysis is to have a sufficient amount of high-quality genomic DNA. Single cells can meet the needs of genetic analysis, and how to obtain enough high-quality genomic DNA from single-cell samples has become a key tec...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 郭兵潘健昌朱珠刘萍
Owner MGI TECH CO LTD
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