Method and kit for quality appraisal for amplification products after single cell genome amplification

A technology for whole-genome amplification and amplification products, which is applied in the field of quality identification of single-cell genome amplification products, can solve problems such as the inability to guarantee the success rate, and achieve the effect of ensuring the success rate and reasonable design

Inactive Publication Date: 2015-07-08
PEKING JABREHOO MED TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, known whole-genome amplification methods include PCR-based methods such as PEP-PCR, DOP-PCR (polymerase chain reaction with degenerate oligonucleotide primers), and multiple strand displacement amplification (MDA, Multiple Displacement Amplification) , are susceptible to interference from m

Method used

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  • Method and kit for quality appraisal for amplification products after single cell genome amplification
  • Method and kit for quality appraisal for amplification products after single cell genome amplification
  • Method and kit for quality appraisal for amplification products after single cell genome amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] 1. Materials:

[0070] 1. Specimen: 5 blastocyst-stage cells biopsied by a cooperative hospital.

[0071] 2. Reagents: Qiagen's whole genome amplification kit, MDA product detection kit, MDA product purification kit, life library construction kit, Agencourt? AMPure? XP magnetic beads, life template preparation kit, life sequencing kit.

[0072] 3. Instruments: PCR instrument, agarose gel electrophoresis system, ion torrent PGM sequencing platform.

[0073] 4. Consumables: 1.5ml, 0.2ml imported centrifuge tubes, Rainin pipettes and pipette tips with filters.

[0074] 2. Operation steps:

[0075] 1. Take cells

[0076] Take 5 cells that have developed to the blastocyst stage, take out the above-mentioned fresh cells under a microscope, and put them into the bottom of a PCR tube containing 1 μl of PBS buffer solution, centrifuge tube number 1.

[0077] 2. Whole genome amplification by MDA method

[0078] Prepare Buffer DLB: Centrifuge the Buffer DLB tube so that the d...

Embodiment 2

[0170] 1. Materials:

[0171] 1. Specimen: 5 blastocyst-stage cells biopsied by a cooperative hospital.

[0172] 2. Reagents: Qiagen's whole genome amplification kit, MDA product detection kit, MDA product purification kit, life library construction kit, Agencourt? AMPure? XP magnetic beads, life template preparation kit, life sequencing kit.

[0173] 3. Instruments: PCR instrument, agarose gel electrophoresis system, ion torrent PGM sequencing platform.

[0174] 4. Consumables: 1.5ml, 0.2ml imported centrifuge tubes, Rainin pipettes and tips with filters.

[0175] 2. Operation steps:

[0176] 1. Take cells

[0177] Take 5 cells that have developed to the blastocyst stage, take out the above-mentioned fresh cells under a microscope, and put them into the bottom of a PCR tube containing 1 μl of PBS buffer solution, centrifuge tube number 2.

[0178] 2. Whole genome amplification by MDA method

[0179] Prepare Buffer DLB: Centrifuge the Buffer DLB tube so that the dry powde...

Embodiment 3

[0271] 1. Materials:

[0272] 1. Specimen: 5 blastocyst-stage cells biopsied by a cooperative hospital.

[0273] 2. Reagents: Qiagen's whole genome amplification kit, MDA product detection kit, MDA product purification kit, life library construction kit, Agencourt? AMPure? XP magnetic beads, life template preparation kit, life sequencing kit.

[0274] 3. Instruments: PCR instrument, agarose gel electrophoresis system, ion torrent PGM sequencing platform.

[0275] 4. Consumables: 1.5ml, 0.2ml imported centrifuge tubes, Rainin pipettes and tips with filters.

[0276] 2. Operation steps:

[0277] 1. Take cells

[0278] Take 5 cells developed to the blastocyst stage, take out the above-mentioned fresh cells under a microscope, and put them into the bottom of a PCR tube containing 1 μl of PBS buffer solution, centrifuge tube number 3.

[0279] 2. Whole genome amplification by MDA method

[0280]Prepare DLB lysate: Centrifuge the DLB lysate so that the dry powder aggregates to ...

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Abstract

The invention relates to a method and a kit for quality appraisal for amplification products after single cell genome amplification. The method comprises the following steps: separating and splitting single cells to obtain the whole-genome DNA of the single cells; carrying out single cell whole-genome amplification on the whole-genome DNA to obtain whole-genome amplification products; placing specific primers in conserved segments on 5 pairs of genomes in an amplification system, carrying out PCR amplification by taking the whole-genome amplification products as a template, carrying out electrophoresis detection on 5 amplification products, judging the quality of the whole-genome amplification products according to a detection result, and showing a result that 3-5 amplification products of uniformly distributed strips on an electrophoretogram are amplification product samples meeting sequencing requirements; constructing a DNA sequencing library on the amplification products meeting the sequencing requirements; carrying out high-throughput sequencing on the DNA sequencing library.

Description

technical field [0001] The invention relates to the field of molecular cell biology, in particular to a method and a kit for identifying the quality of a single-cell genome amplification product. Background technique [0002] Studying DNA at the single-cell level can effectively avoid many limitations of multi-cell level research, especially in the field of embryos. Due to the chromosomal testing of early embryos, DNA analysis at the single-cell level is necessary. Therefore, the development of technical methods applied to the study of single cells in order to reveal the rules of cell heterogeneity is of great significance for better cell biology research. In single-cell analysis, studies of single-cell genomes, expression profiles, and metabolomes are included. At present, trace DNA and single-cell genome research have been widely used in archaeology, microbial ecology, medical detection, forensic detection, clinical diagnosis and other fields. In reproductive medicine, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 冯涛王丹红
Owner PEKING JABREHOO MED TECH CO LTD
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