Reagent kit for simultaneously detecting multiple deafness genes on single cell level

A deafness gene and single-cell technology, applied in the field of PCR amplification, can solve the problems of long amplification time, high amplification cost, and low quality of amplification products.

Active Publication Date: 2015-12-30
ZHEJIANG ANNOROAD BIO TECH CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] (2) GJB3 gene mutation can lead to dominant or recessive hereditary non-syndromic deafness
Therefore, the amplification of multiple deafness genes needs to be carried out in multiple PCR reaction procedures, which has the problems of long amplification time and high amplification cost.
In addition, if multiple genes are amplified under the conditions of the same PCR reaction program, there is often the problem that the quality of the amplified product of one or more genes is not high.

Method used

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  • Reagent kit for simultaneously detecting multiple deafness genes on single cell level
  • Reagent kit for simultaneously detecting multiple deafness genes on single cell level
  • Reagent kit for simultaneously detecting multiple deafness genes on single cell level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1: MDA method amplifies whole genome DNA of human single cell

[0091] Using the QIAGENREPLI-gSingleCellKit Single Cell Whole Genome Amplification Kit, the MDA method was used to amplify whole genome DNA from human single cell samples.

[0092] Human single cells were collected in a volume within 0.5 μL into 0.2 mL thin-walled PCR tubes containing 4 μL of PBSsc1x (clearlid) provided in the kit.

[0093] Prepare BufferD2 (12 reactions), the components are as follows:

[0094]

[0095] The prepared BufferD2 can be used for 12 reactions. For each sample, add 3 μL BufferD2 to the PCR tube and mix well. Perform the reaction on a PCR instrument, react at 65°C for 10 minutes, add 3 μL StopSolution to the PCR tube, mix well, and place on ice.

[0096] Prepare MasterMix with the following ingredients:

[0097]

[0098] Add the prepared MasterMix into the PCR tube in 2.2.1.2 and mix evenly, and react on the PCR instrument. The reaction procedure is as follows:...

Embodiment 2

[0101] Example 2 Using the same PCR reaction program conditions to simultaneously amplify 20 sites of 4 deafness genes

[0102] A total of 10 pairs of primers were designed for hereditary non-syndromic deafness genes GJB2, GJB3, 12SrRNA, and SLC26A4, covering 20 mutation sites of 4 genes. The primer information is as follows:

[0103] Table 1 Primer sequence information

[0104]

[0105] The amplified human single-cell whole-genome DNA sample was taken as a template for PCR reaction, and the sample was subjected to PCR amplification with 10 pairs of primers designed above, a total of 10 reactions, but the same PCR reaction procedure was used. Each reaction requires 25ng template DNA, and 10 reactions require a total of 250ng template DNA.

[0106] The PCR reaction system is as follows:

[0107]

[0108] The PCR reaction program was as follows: 95°C for 15min, (95°C for 30s, 59°C for 30s, 72°C for 1min)×40 cycles, 72°C for 10min, and 4°C for storage.

[0109] 1% agaro...

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Abstract

The invention provides a reagent kit for simultaneously detecting multiple deafness genes on a single cell level. The invention also provides a method for simultaneously detecting various deafness gene sites on a single cell level. By adopting a single-cell whole genome amplification technology, a sample type with little content of DNA such as a single-cell sample is effectively amplified to whole-genome DNA with total amount of micro-gram by virtue of MDA (multiple displacement amplification). The obtained DNA is amplified by virtue of PCR of a specific primer, Sanger sequencing is carried out for an amplified product on a sequencing instrument, and various genetic deafness genes can be effectively detected.

Description

technical field [0001] The invention relates to a PCR amplification method. Specifically, the present invention relates to a PCR amplification method capable of simultaneously amplifying multiple deafness genes, especially deafness genes derived from human single-cell whole genome. In addition, the present invention also provides a kit for simultaneously detecting multiple deafness genes at the single cell level. Background technique [0002] Deafness is a clinically common disease, about 60% of which are caused by genetic factors. According to the second sample survey of disabled people in 2006, the total number of disabled people in my country is 80 million, of which 26.7 million are hearing disabled, and there are about 800,000 hearing-impaired children aged 1-7. Among the 20 million newborns in my country every year, the incidence of severe hearing impairment is 1‰~3‰. The birth hearing defect of newborns has brought serious economic and spiritual burdens to families ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 柳青白金蕾玄兆伶李大为梁峻彬陈重建
Owner ZHEJIANG ANNOROAD BIO TECH CO LTD
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