DNA labels, PCR primers and application thereof

A label and mixture technology, which is applied in DNA label, PCR primer and its application field, can solve the problems that the deafness gene detection technology needs to be improved.

Active Publication Date: 2014-11-05
海南华大基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current deafness genetic ...

Method used

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  • DNA labels, PCR primers and application thereof
  • DNA labels, PCR primers and application thereof
  • DNA labels, PCR primers and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] According to the method of determining whether there is a mutation in the deafness gene of multiple DNA samples according to the present invention, the deafness gene detection is carried out, specifically as follows:

[0079] (1) Primer design

[0080] According to the CDS region and 12S rRNA of GJB2, GJB3 and SLC26A4 genes, the primer design software was used to design the amplification primers of the above gene loci. The length of the amplification primers was about 20 bases. Use software to design and evaluate the DNA index (index) sequence. The index sequence contains 7 bases. When designing the index sequence, it should avoid sequences with high similarity to the sequencing primers and the tagged PCR amplification primers to form a hairpin structure or dimerization. Body and other secondary structures. Refer to Table 1 for the amplification primers designed by the present invention for the above-mentioned GJB2 and GJB3 gene CDS regions, SLC26A4 gene No. 2-21 exon ...

Embodiment 2

[0164] According to the method of determining whether there is a mutation in the deafness gene of multiple DNA samples according to the present invention, the deafness gene detection is carried out, specifically as follows:

[0165] (1) For samples 1-5 in Example 1, repeat the experimental operations of primer design, PCR amplification and amplified product mixing, end repair, adapter ligation, and library detection in Example 1, and also obtain the samples in Example 1. Library samples of known concentration, for later use.

[0166] (2) Preparation of Ion PGM TM Sequencing template

[0167] ①One Touch TM 2

[0168] Detect the library concentration according to the Agilent Bioanalyzer2100, dilute the library to 80pM, prepare the OneTouch 2 reaction mixture according to Table 8, transfer the adapter and load it into the initialized OneTouch TM 2. See OneTouch for details on the operation process TM manual.

[0169] Amplification solution, respectively, 1000 μL of amplifi...

Embodiment 3

[0181] According to the method of determining whether there is a mutation in the deafness gene of multiple DNA samples according to the present invention, the deafness gene detection is carried out, specifically as follows:

[0182] (1) Primer design: According to the selected 20 deafness gene mutation sites to be detected and / or typed, they are: 35delG, 167delT, 176-191del16, 299_300delAT and 235delC of the GJB2 gene, 538C>T of the GJB3 gene 547G>A, 281C>T, 589G>A, IVS7-2A>G, 1174A>T, 1226G>A, 1229C>T, 1975G>C, 2027T>A, 2162C>T, 2168A>G and IVS15+5G>A, 1494C>T and 1555A>G of 12S rRNA gene. The amplification primers designed by the present invention for the 20 mutation sites of the above four deafness genes are selected from the primers in Example 1, see Table 15.

[0183] Table 15

[0184]

[0185]

[0186] (2) In addition to the different samples tested, subsequent experimental steps (ie: PCR amplification and amplification product mixing, end repair, adapter ligatio...

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Abstract

The invention discloses DNA labels, PCR primers and application thereof. A group of the DNA labels are composed of nucleotides as shown in SEQ ID No. 79 to 129. The group of the DNA labels can be used for establishment of a nucleic acid sequencing library to realize accurate differentiation of the nucleic acid sequencing library. The sources of DNA samples can be accurately characterized by connecting the DNA labels with DNA or equivalents thereof.

Description

technical field [0001] The present invention relates to the technical field of deafness gene detection, in particular, to DNA tags, PCR primers and applications thereof, more specifically, to a set of isolated DNA tags, a set of isolated PCR primers, a method for constructing a nucleic acid sequencing library, A nucleic acid sequencing library, a method for determining whether there is a mutation in a DNA sample deafness gene, a method for determining whether a variety of DNA samples have a mutation in a deaf gene, and a kit for determining whether a DNA sample has a mutation in a deafness gene. Background technique [0002] Deafness is one of the common human diseases. According to the results of the second national sample survey of disabled people at the end of 2006, there are 26.7 million people with hearing disabilities in my country, accounting for 19.3% of the total number of disabled people. There are 800,000 hearing-impaired children aged 1-7, and more than 30,000 de...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C40B50/06
Inventor 冯大飞邹婧欧日晶侯强张俊青程秀易鑫
Owner 海南华大基因科技有限公司
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