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

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...

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.

Images