Specific detection primers and detection liquid phase chip for ADH1B gene mutation

A detection solution and specific technology, applied in the field of molecular biology, can solve the problems of inconvenient judgment, low detection sensitivity, narrow analysis range, etc., and achieve the effect of avoiding uncertain factors, low cross-reaction rate, and avoiding cross-reaction

Inactive Publication Date: 2014-02-12
SUREXAM BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, ADH1B gene mutation detection methods mainly include: matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), creation of restriction site PCR (CRS-PCR), microarray chip technology, matrix-assisted laser desorption ionization Time-of-flight mass spectrometry is a soft ionization technology that has powerful and mature functions in the detection of protein and other biological macromolecules. However, in the field of nucleic acid detection, due to the particularity of nucleic acid molecules, the detection is subject to certain restrictions.
The choice of primers for creating restriction sites is small, and only the adjacent sequences of polymorphic sites can be used, and after digestion of CRS-PCR products, the difference in length of fragments between different alleles is only about one primer length (generally 20bp~25bp), if the amplified length is relatively large, such a small fragment length difference is not easy to be displayed during electrophoresis, which will bring great inconvenience to the genotype judgment
However, microarray technology has problems such as high cost, complexity, low detection sensitivity, poor repeatability, narrow analysis range, complex synthesis and immobilization of probes, etc., so these detection methods are difficult to meet the needs of practical applications.

Method used

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  • Specific detection primers and detection liquid phase chip for ADH1B gene mutation
  • Specific detection primers and detection liquid phase chip for ADH1B gene mutation
  • Specific detection primers and detection liquid phase chip for ADH1B gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1 ADH1B gene mutation detection liquid chip mainly includes:

[0021] 1. ASPE Primers

[0022] Specific primer sequences were designed for wild type and mutant types of six common genotypes of ADH1B gene A3170G, G3641T, G4564T, A5998G, G531A and T4973C. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0023] Table 1 ASPE primer sequence of ADH1B gene (tag sequence + specific primer sequence)

[0024]

[0025]

[0026] 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 100 pmol / mL stock solution with 10 mmol / L Tris Buffer.

[0027] 2. Microspheres coated with ant...

Embodiment 2

[0038] Example 2 Detection of samples using the ADH1B gene mutation detection liquid chip described in Example 1

[0039] The formula of described various solutions is as follows:

[0040] 50mM MES buffer (pH5.0) formula (250ml):

[0041]

[0042] 2×Tm hybridization buffer

[0043]

[0044] Store at 4°C after filtration.

[0045] ExoSAP-IT kit was purchased from US USB Company.

[0046] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0047] 1. Sample DNA extraction:

[0048] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0049] 2. PCR amplification of samples to be tested

[0050] Design 6 pairs of primers and multiplex PCR to amplify 6 target sequences containing six common genotypes of ADH1B gene A3170G, G3641T, G4564T, A5998G, G531A and T4973C in one step, and the product sizes are 397bp, 349bp, 306bp, 323bp, 227bp , 248bp, and the primer sequences (...

Embodiment 3

[0096] The liquid phase chip of embodiment 3 different ASPE primers is to the detection of ADH1B gene SNP site

[0097] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0098] Taking the ADH1B gene A3170G, G3641T, A5998G and T4973C 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 A3170G, G3641T, A5998G and T4973C, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQ ID NO.1-SEQ ID NO.12. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.25-SEQ ID NO.36. The specific design is shown in the following table (Table 8). 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.

[0099] Tab...

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Abstract

The present invention discloses a detection liquid phase chip and specific primers for ADH1B gene mutation. The liquid phase chip mainly comprises: ASPE primers comprising a 5' terminal tag sequence and a 3' terminal target gene mutational site-targeted specific primer sequence, wherein the specific primer sequence comprises A3170G site-targeted SEQ ID NO.13, A3170G site-targeted SEQ ID NO.14, G3641T site-targeted SEQ ID NO.15, G3641T site-targeted SEQ ID NO.16, G4564T site-targeted SEQ ID NO.17, G4564T site-targeted SEQ ID NO.18, A5998G site-targeted SEQ ID NO.19, A5998G site-targeted SEQ ID NO.20, G531A site-targeted SEQ ID NO.21, G531A site-targeted SEQ ID NO.22, and / or T4973C site-targeted SEQ ID NO.23 and T4973C site-targeted SEQ ID NO.24; anti-tag sequence coated microspheres; and amplification primers. According to the present invention, coincidence frequency of the detection results of the detection liquid phase chip and the sequencing method is up to 100%, and single and parallel detection on the wild-type with multiple mutational sites and the mutant-type with multiple mutational sites can be achieved.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for ADH1B gene mutation detection and a liquid phase chip. Background technique [0002] The ADH1B gene is called alcohol dehydrogenase 1B, and its English name is alcohol dehydrogenase-1B. It is located on the long arm of chromosome 4 4q23, between 100227526 and 100242571 base pairs. The protein encoded by the ADH1B gene is a member of the alcohol dehydrogenase family, which can digest and metabolize many substances, including ethanol, retinol, fatty alcohols, dehydrogenated sterols, and lipid peroxidation products. This encoded protein consists of homo- and heterodimers of several α, β and γ subunits, is highly active in ethanol oxidation and plays an important role in ethanol metabolism. Recently, it has also been found that mutations in the ADH1B gene are associated with cancer, liver dysfunction, coronary heart diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q1/6886C12Q2600/156
Inventor 许嘉森何嘉英
Owner SUREXAM BIO TECH
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