Kit and method for detecting mutant alpha-Mediterranean anemia genes through HRM (high resolution melting) method

Abstract

The invention relates to a disease gene detection technology, and particularly relates to a kit and method for detecting mutant alpha-Mediterranean anemia genes through an HRM (high resolution melting) method. The kit provided by the invention comprises two PCR (polymerase chain reaction) tubes, namely an alpha-PCR tube 1 and an alpha-PCR tube 2, wherein each PCR tube contains a PCR reagent; the PCR reagent comprises primers, a PCR Buffer, dNTPs (deoxyribonucleotide triphosphate), MgCl2, DNA (deoxyribonucleic acid) polymerase and saturated fluorescent dyes. By using the optimized PCR-HRM reaction conditions, 6 mutant alpha-Mediterranean anemia genes, including one of alpha-cod30, alpha-cod31, alpha-cod59, alpha-WM-cod122, alpha-QS-cod125 and alpha-CS-cod142 or a combination of more than one, can be simultaneously detected.

Description

technical field [0001] The invention relates to a disease gene detection technology, in particular to a kit and a method for detecting mutant α-thalassemia genes by the HRM method. Background technique [0002] High Resolution Melting (HRM) technology is a new genetic analysis method for mutation scanning and genotyping that has emerged in recent years. HRM is not limited by the site and type of mutated bases, and does not require sequence-specific probes. After PCR, high-resolution melting can be run directly to complete the analysis of sample mutations, single nucleotide polymorphisms, -SNPs, and methylation. , HLA matching, etc. analysis. Compared with other genetic typing techniques, this method is not limited by the site and type of mutated bases, is easy to operate, has the advantages of high sensitivity, good specificity, low cost, rapid, high-throughput detection, accurate results, and A true closed-tube operation is realized. [0003] The HRM principle is based o...

AI Technical Summary

Problems solved by technology

The RFLP method is a method that combines PCR with restriction endonucleases, but this method has the following disadvantages: the test operation is cumbersome, the detection period is long, the cost is high, and there are false positives caused by incomplete first-round enzyme digestion

However, this method also has many disadvantages: the equipment and reagents are expensive, and it is not suitable to directly detect mutations by sequencing for some genes with long fragments or many exons; in addition, it is not suitable for the detection of a large number of clinical samples , Missing samples, and mixed samples can’t get a good sequencing map. Accurate data must be obtained through cloning and sequencing, and multiple experiments; for samples with GC enrichment and difficult templates, it is also difficult to obtain accurate data through one-time sequencing.

These patents are difficult to commercialize because they all have common deficiencies: their probe design requires a large number of probe screening to obtain satisfactory results; when the target gene is amplified, it is generally divided into 2-3 sections, The fragment length is about 300bp-600bp. During the hybridization process, the longer the fragment length, the lower the hybridization efficiency. Especially for gene chips with glass slides as carriers, the empirical value is no more than 400bp. In order to compensate for the hybridization efficiency Insufficient, the corresponding measure is to extend the hybridization time or even overnight hybridization, but this measure is not suitable for the original intention of rapid detection; the gene chip detection method with membrane or chip as the carrier has a complicated detection process, and is designed to detect PCR products The operation of the open tube is very easy to cause pollution

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