Method of direct DNA sequencing for screening hepatolenticular degeneration disease gene mutation site through PCR amplification

Abstract

A method of direct DNA sequencing for screening a hepatolenticular degeneration disease gene mutation site through PCR amplification. The invention relates to a method of direct DNA sequencing after the PCR amplification for screening a disease mutation site of ATP7B gene, thereby providing the basis for accurately diagnosis and prenatal diagnosis of the hepatolenticular degeneration disease. The method includes the processes of primer design, genome DNA extraction, PCR amplification, PCR product purification, PCR product sequencing and sequence comparative analysis and the like, wherein the key to decide effectiveness of the method is specificity and sensitivity of the primer. The primer in the invention is strong in specificity and high in sensitivity and is wide in coverage. The method can detect the all 21 exon regions, promoter regions and partial intron regions of the ATP7B gene, and can provide firm foundation of clinical diagnosis application of the hepatolenticular degeneration through genetic detection.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for determining mutation sites of ATP7B genes by direct sequencing of polymerase chain reaction (PCR) products. technical background [0002] Hepatolenticular degeneration, also known as Wilson's Disease (WD), is an autosomal recessive genetic disorder of copper metabolism. Foreign epidemiological survey data show that the prevalence rate is 1.5-3.0 / 100,000, the gene frequency is 0.3%-0.7%, and the heterozygote frequency is estimated to be 1 / 100. There is no large epidemiological report of this disease in my country. According to related reports, Wilson's disease ranks second among all monogenic diseases. [0003] The diagnosis of Wilson disease is often judged by indicators such as plasma ceruloplasmin, blood copper, urine copper content, K-F ring, etc., but only 80% of WD patients show low plasma ceruloplasmin, and 30-40% of patients have K-F ring appear...

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a low-cost, simple and fast, high-accuracy, and highly operable gene detection method for Wilson disease, reduce the probability of missed diagnosis and misdiagnosis due to inconspicuous clinical symptoms, and at the same time It can assist prenatal screening and guide prenatal and postnatal care

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