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Nucleic acid sequence for detecting deaf genes and applications thereof

A deafness gene and nucleotide sequence technology, applied in the field of gene sequencing and biological testing, can solve the problems of time-consuming, laborious, high cost, and difficulty in detecting different mutation sites of multiple genes at the same time, and achieve low-cost effects

Active Publication Date: 2016-09-14
成都凡迪医疗器械有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these techniques are either time-consuming or labor-intensive, or have high costs, or are difficult to detect different mutation sites of multiple genes at the same time, or can only be used to detect known mutations and cannot cover new mutations or low-frequency mutations

Method used

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  • Nucleic acid sequence for detecting deaf genes and applications thereof
  • Nucleic acid sequence for detecting deaf genes and applications thereof
  • Nucleic acid sequence for detecting deaf genes and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Extracting DNA from Whole Blood Samples

[0067] 1) Take the sample out of the refrigerator at -20°C, dissolve it at room temperature, and turn on the water bath and set it to 56°C.

[0068] 2) Add 20ul of proteinase K to a 1.5ml centrifuge tube, add 200ul of fully dissolved whole blood (mix by inverting before use), shake and mix, and centrifuge for a short time to remove droplets on the inner wall of the tube.

[0069] 3) Add 200 ul buffer solution GB, vortex to mix, and centrifuge for a short time to remove the liquid droplets on the inner wall of the tube. Put the centrifuge tube in a water bath at 56° C. for 10 min, and shake the sample gently from time to time.

[0070] 4) Take the centrifuge tube out of the water bath, let it cool slightly, add 200ul absolute ethanol, shake and mix well, centrifuge for a short time and let it stand at room temperature for 3 minutes.

[0071] 5) Transfer the above mixed solution into the adsorption column CR2. Care sho...

Embodiment 2

[0079] Embodiment 2: multiplex PCR amplification

[0080] Using the DNA obtained in Example 1 as a template, multiplex PCR amplification was performed.

[0081] The multiplex PCR reaction system described therein is a 25ul system, and the specific reaction system and reaction procedures are shown in Table 4 and Table 5. The "multiple PCR primers" are primer pools, see Table 1 and Table 2.

[0082] Table 4: Reaction system of primer pool ID No: 1-primer pool ID No: 4

[0083] serial number

Reagent

Volume (ul)

1

template DNA

2

2

rTaq polymerase

0.2

3

10X PCR buffer

2.5

4

dNTP Mixture (2.5mM)

2

5

Multiplex PCR Primers (100uM)

4

6

Sterile distilled water

14.3

Total

25

[0084] Table 5: Reaction system of primer pool ID No: 5-primer pool ID No: 8

[0085] serial number

Reagent

Volume (ul)

1

template DNA

2

2

LA Taq polymerase

...

Embodiment 3

[0089] Example 3: Library Preparation

[0090] The PCR products obtained in Example 2 were mixed and purified using Agencourt AMPure XP-Nucleic Acid Purification Kit. The purified product was prepared for library preparation, and the library preparation steps were as follows:

[0091] 1. End repair

[0092] Table 7: End repair reaction system

[0093]

[0094] After the reaction system was shaken and centrifuged, it was incubated at room temperature for 20 min. After incubation, use AgencourtAMPure XP-Nucleic Acid Purification Kit for purification, and finally dissolve in 27ul nuclease-free water.

[0095] 2. Joint connection

[0096] Table 8: Linker Ligation Reaction System

[0097]

[0098] After the adapter ligation reaction mixture was shaken and centrifuged, it was placed on a PCR machine and incubated at 25°C for 15 minutes and at 72°C for 5 minutes. After incubation, use Agencourt AMPure XP-Nucleic Acid Purification Kit for purification, and finally dissolve...

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Abstract

The invention discloses a nucleic acid sequence for detecting the deaf genes and applications thereof, and belongs to the fields of gene sequencing and biological examination. A set of specific primers for detecting the deaf genes comprises nucleic acid sequences represented by SEQ ID No.1 to SEQ ID No.17. A novel specific multiple PCR primer, which comprises whole exon with a total length of 9497 bp and is capable of detecting four deaf genes at the same time, is designed. Furthermore, a method using the specific multiple PCR primer to carry out detection is also provided. Compared with the prior art, a high-throughput sequencing technology is adopted to detect the deaf genes, and all mutation sites on four deaf genes can be rapidly and accurately detected, including the hot spot mutations and new mutations. The provided detection method has the characteristics of high throughput, low cost, and high detection rate.

Description

technical field [0001] The invention relates to a nucleic acid sequence for detecting deafness genes and its application, and belongs to the fields of gene sequencing and biological testing. Specifically, it is a detection method for detecting the whole exons of four deafness genes, and the specific primers used in the method, especially a method involving multiplex PCR technology combined with high-throughput sequencing technology for detection. Background technique [0002] Deafness is one of the most common sensory system diseases, the incidence rate in newborns is about 1 / 1000, and about 60% of deafness patients are caused by genetic factors. According to different phenotypes, it is divided into syndromic deafness (SHI) and non-syndromic deafness (NSHI). According to different ways of inheritance, hereditary deafness is divided into five types: autosomal dominant (AD), autosomal recessive (AR), X-linked, Y-linked, and mitochondrial. Among them, autosomal recessive The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q2600/158C12Q2600/16C12Q2600/178C12Q2531/113C12Q2525/191C12Q2537/143
Inventor 杨光辉岑忠崔书建
Owner 成都凡迪医疗器械有限公司
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