Method and oligonucleotide for detecting δf508 mutation of cftr gene

Abstract

The invention discloses a method for detecting mutation on the locus deltaF508 of a CFTR (Cystic Fibrosis Transmembrane Regulator) gene and an oligonucleotide, relating to a pair of specific amplification primers SEQ NO 1 and SEQ NO 2 and a pair of specific detection probes SEQ NO 3 and SEQ NO4. The method has the advantages of short detection period, high specificity, high accuracy, high sensitivity, low conditional dependency, low pollution risk and the like, and can be used for assisting in accurately and rapidly diagnosing cystic fibrosis and male infertility.

Description

technical field [0001] The invention belongs to the field of gene mutation detection, and in particular relates to an oligonucleotide and a method for highly sensitive detection of CFTR gene ΔF508 site mutation. Background technique [0002] Cystic fibrosis (CF) is the most common fatal autosomal recessive genetic disease in Caucasians, and its incidence is higher in Western European, Northern European and North American populations, accounting for about 1 / 2500 live births , the incidence rate in Asians is low, about 1 / 100,000. One of the typical clinical manifestations of CF is male patients with congenital absence of bilateral or unilateral vas deferens, resulting in infertility. Congenital absence of the vas deferens (CAVD) is an important cause of posttesticular factor azoospermia and male infertility, and its pathogenesis is related to CF gene mutation and mesonephric duct development defects. [0003] In 1989, Rommens et al positionally cloned and identified the gene...

AI Technical Summary

Problems solved by technology

Among them, SYBR Green I is not as specific as the double-hybrid probe method and Taqman probe method because it is an unsaturated dye, and its specificity must be judged by observing the melting curve. In addition, it cannot recognize a specific double-stranded DNA sequence, as long as it is double-stranded DNA The sequence will be combined with luminescence, so the background is usually high, and false positives may occur when used clinically

The two-hybridization probe method involves two specific probes that are complementary to the template and adjacent (distance 1-5bp), the 3' end of the upstream probe is labeled with a donor fluorophore, and the 5' end of the adjacent downstream probe is End-labeled Red640 acceptor fluorophore, the cost is relatively expensive

The molecular beacon method involves a stem-loop double-labeled oligonucleotide probe in a hairpin structure. The nucleic acid sequences at both ends of the probe are complementary paired, and a fluorescent group labeled at one end is labeled with a quencher group at the other end. The biggest disadvantage of this method is that the probe cannot be completely combined with the template during hybridization, so the stability is poor, and the labeling process is more complicated when the probe is synthesized.

The Taqman probe method involves a gene-specific probe (usually 20-30bp long) complementary to the template. The 5' end and the 3' end of the probe are respectively labeled with a reporter fluorophore and a quencher fluorophore, but The longer probe sequence in this method makes the distance between the reporter fluorophore and the quencher fluorophore at both ends of the probe farther away, resulting in incomplete fluorescence quenching, and the quencher group will also produce fluorescence of different wavelengths. This will make the background higher

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