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Combined primer for screening human deafness gene mutation and application of combined primer

A human, screening technology, applied in recombinant DNA technology, microbial assay/examination, biochemical equipment and methods, etc., can solve problems such as irreversible and severe hearing loss, and achieve high-throughput effects

Active Publication Date: 2015-04-29
南京普东兴生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A single dose can cause profound and irreversible hearing loss in individuals with this mutation

Method used

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  • Combined primer for screening human deafness gene mutation and application of combined primer
  • Combined primer for screening human deafness gene mutation and application of combined primer
  • Combined primer for screening human deafness gene mutation and application of combined primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Sample Collection and DNA Extraction

[0059] Note: In order to ensure that the sample is not contaminated by food or drink, do not eat or drink within 30 minutes before sampling.

[0060] 1) Please rinse your mouth with water before collection.

[0061] 2) Open the package of the oral swab package, swallow first, then insert a clean oral swab into the subject's mouth, and scrape repeatedly 10 times against the inside of the right cheek.

[0062] 3) Take out the swab, put it face up into the sponge card slot, and let it dry naturally for 30 minutes.

[0063] 4) Seal the sponge card slot with sealing tape, and it can be stored at room temperature for 1-2 weeks.

[0064] 5) Use Tiangen Oral Swab Genome Extraction Kit DP322 to extract DNA from the sample.

[0065] The extraction results of 10 subjects' cavity swabs were 53.4, 48.5, 49.6, 42.6, 56.7, 58.5, 46.3, 50.4, 41.2 and 44.4 ng / μl.

Embodiment 2

[0066] Embodiment 2 PCR amplification

[0067] Each primer was mixed to a final concentration of 2 μM, and the amplification reaction was carried out according to the following reaction system and amplification procedure.

[0068] The multiplex PCR system is as follows:

[0069]

[0070] The multiplex PCR reaction conditions are as follows:

[0071]

[0072] Table 1: A label (Barcode) sequence for human deafness gene mutation screening:

[0073] label number

sequence

SEQ ID NO:

BC001

TGAACGTG

1

BC002

TCATGTCC

2

BC003

TGTGAGGT

3

BC004

GAGTGAAC

4

BC005

TGGATCAG

5

BC006

CTCTCCTA

6

BC007

TAAGGAGC

7

BC008

CGTAAGTG

8

BC009

CCATCTTC

9

BC010

TACA AGGC

10

BC011

GTCTTGAC

11

BC012

TCTGGATC

12

BC013

GCAGTAGA

13

BC014

TAAGGTCG

14

BC015

TTCCAAGC

...

Embodiment 3

[0081] Example 3: Construction of PCR product purification and sequencing library

[0082] Use Agencourt AMPure XP magnetic beads from Beckman Company to purify the PCR product, and perform quality control on the amplified library. The quality control method is as follows: first use Nanodrop 2000 for preliminary quantification of the library, and mix it in equal proportions according to the molar amount; use Qubit 2.0 Perform final quantification of the pooled library as a dilution standard for the final emulsion PCR.

[0083] The final concentrations of the 10 libraries obtained were 5.5, 4.2, 5.0, 5.2, 4.8, 4.6, 4.2, 5.8, 5.3 and 4.8 ng / μl, respectively.

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Abstract

The invention discloses a combined primer for screening human deafness gene mutation. The combined primer comprises a combined forward primer and a combined reverse primer, wherein the combined forward primer consists of two parts namely a joint part P1 sequence and a forward primer sequence, and the combined reverse primer sequentially consists of a joint part P2 sequence, a label sequence and a reverse primer sequence. The invention also discloses a kit for screening the human deafness gene mutation. The invention also discloses applications of the combined primer and the kit in preparation of a reagent for screening the human deafness multi-gene mutation. A primer containing the label (Barcode) sequence is used for differentiating different samples, and at least 32 samples can be detected by performing a method provided by the invention once, so that combined primer can be widely applied to general investigation of deafness susceptible genes. The price of the combined primer disclosed by the invention is far lower than that of imported instruments or reagents, so that the sequencing cost is greatly reduced and then the detection cost is also greatly reduced.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a combined primer for screening human deafness gene mutations and an application thereof. Background technique [0002] Deafness is one of the most common sensory impairment diseases, and congenital deafness can cause speech impairment. According to the second national sampling survey on disabled people, the number of hearing disabled people in my country is about 20.04 million, accounting for 24.16% of the total number of disabled people; about 1.27 million people are speech disabled due to deafness, accounting for 1.53% of the total number of disabled people. In 2010, an estimated 278 million people worldwide were hearing impaired. [0003] The influencing factors of deafness are mainly divided into environmental factors and genetic factors. With the reduction of the incidence of deafness caused by environmental factors and the improvement of disease diagnosis and treatment, gene...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q2600/156C12Q2600/16C12Q2537/143C12Q2563/159C12Q2563/185
Inventor 戴琳超叶珊陆祖宏
Owner 南京普东兴生物科技有限公司
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