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Specific primer and liquid phase chip for detecting TYR gene mutation

A detection solution and specificity technology, applied in the field of molecular biology, can solve the problems of difficult to meet the needs of practical applications, low degree of automation, high false positive rate, etc., to avoid uncertain factors, simple steps, good detection specificity Effect

Active Publication Date: 2014-06-11
SUREXAM BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Currently, TYR gene mutation detection methods mainly include: Illumina fiber optic bead chip technology, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and fluorescent quantitative PCR technology, although Illumina fiber optic bead chip technology is highly sensitive and Accurate high-throughput detection system, but with low degree of automation and many manual operations, it is difficult to meet the needs of practical applications. Fluorescence quantitative PCR technology has the characteristics of high sensitivity, strong specificity, and high degree of automation, but there are also samples that are easy to contaminate , high false positive rate, and can only detect one type of mutation each time, which also cannot meet the needs of practical applications. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a soft ionization technology, which is used in the detection of biological macromolecules such as proteins. There are powerful and mature functions in detection, but in the field of nucleic acid detection, due to the particularity of nucleic acid molecules, detection is limited to a certain extent, and it is difficult to meet the needs of practical applications

Method used

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  • Specific primer and liquid phase chip for detecting TYR gene mutation
  • Specific primer and liquid phase chip for detecting TYR gene mutation
  • Specific primer and liquid phase chip for detecting TYR gene mutation

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1 TYR gene mutation detection liquid chip mainly includes:

[0022] 1. ASPE Primers

[0023] Specific primer sequences were designed for wild-type and mutant types of four common genotypes of TYR gene, G6895A, C1205T, C575A and A99214C. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0024] Table 1 ASPE primer sequence of TYR gene (tag sequence + specific primer sequence)

[0025]

[0026]

[0027] Each ASPE primer consists of two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in Table 1 above). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0028] 2. Microspheres coated with anti-tag sequences ...

Embodiment 2

[0039] Example 2 Detection of samples using the TYR gene mutation detection liquid chip described in Example 1

[0040] The formula of described various solutions is as follows:

[0041] 50mM MES buffer (pH5.0) formula (250ml):

[0042]

[0043] 2×Tm hybridization buffer

[0044]

[0045] Store at 4°C after filtration.

[0046] ExoSAP-IT kit was purchased from US USB Company.

[0047] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0048] 1. Sample DNA extraction:

[0049] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0050] 2. PCR amplification of samples to be tested

[0051] 4 pairs of primers were designed, and multiplex PCR amplified 4 target sequences containing four common genotypes of TYR gene G6895A and C1205T, C575A and A99214C in one step. The product sizes were 307bp, 355bp, 408bp and 269bp respectively. NO.25-32) see Table 3 above.

[005...

Embodiment 3

[0094] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of TYR gene SNP site

[0095] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0096]Taking TYR gene G6895A, C1205T, C575A and A99214C site mutation detection liquid chip as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of G6895A, C1205T, C575A and A99214C, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQ ID NO.1-SEQ ID NO.8. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.17-SEQ ID NO.24. The specific design is shown in the following table (Table 7). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0097] Table 7 Desig...

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Abstract

The invention discloses a liquid phase chip and a specific primer for detecting TYR gene mutation. The liquid phase chip mainly comprises: ASPE primers, microspheres coated by different anti-tag sequences, and an amplification primer, wherein each ASPE primer is composed of a tag sequence at a 5' end and specific primer sequences at a 3' end and aiming at target gene mutation sites, and the specific primer sequences are as follows: SEQ ID NO.9 and SEQ ID NO.10 aiming at a G6895A site, SEQ ID NO.11 and SEQ ID NO.12 aiming at an C1205T site, SEQ ID NO.13 and SEQ ID NO.14 aiming at a C575A site, and / or SEQ ID NO.15 and SEQ ID NO.16 aiming at an A99214C site. The coincidence rate of the detection result of the detection liquid phase chip disclosed by the invention with that of a sequencing method reaches as high as 100%, and parallel detection of a wild type and a mutant of a plurality of mutation sites is achieved.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a TYR gene mutation detection specific primer and a liquid phase chip. Background technique [0002] Tyrosinase (TYR) is located on the long arm of chromosome 11 11q14.3, specifically between 88911039 and 89028926 base pairs on chromosome 11. Tyrosinase (TYR) is a 75kD copper-containing enzyme derived from embryonic neural crest cells and is a key enzyme in melanin metabolism and catecholamines. Studies have found that more than 100 mutations of the TYR gene can be found in patients with oculocutaneous albinism type I. These mutations disrupt the normal production of melanin, thereby reducing the coloration of hair, skin and eyes. In addition, tyrosinase may be an important antigen of vitiligo autoimmunity. It has recently been found that some patients with vitiligo have tyrosinase antibodies in their serum, which are closely related to th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B40/06C12Q1/68C12N15/11
Inventor 吴诗扬陈昌华
Owner SUREXAM BIO TECH
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