Method for detecting PAH (Polycyclic Aromatic Hydrocarbon) gene mutation by utilizing high-resolution melting curve analysis technique

A high-resolution melting and curve analysis technology is applied in the field of detection of phenylalanine hydroxylase gene mutation, which can solve the problems of inappropriate detection, high cost, time-consuming and labor-intensive, and achieve fast detection speed, low cost, and avoidance of crossover. The effect of pollution

Inactive Publication Date: 2014-01-15
上海中优医药高科技股份有限公司
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Problems solved by technology

[0004] At present, the commonly used mutation detection method is direct DNA sequencing method. PCR products can be directly analyzed by DNA sequence, which ca

Method used

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  • Method for detecting PAH (Polycyclic Aromatic Hydrocarbon) gene mutation by utilizing high-resolution melting curve analysis technique
  • Method for detecting PAH (Polycyclic Aromatic Hydrocarbon) gene mutation by utilizing high-resolution melting curve analysis technique
  • Method for detecting PAH (Polycyclic Aromatic Hydrocarbon) gene mutation by utilizing high-resolution melting curve analysis technique

Examples

Experimental program
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Embodiment

[0034] Example Taking the detection of exon 7 of the PAH gene as an example

[0035] 1. Using the silica gel adsorption method to extract the genomic DNA of the oral epithelial cells of 15 subjects, the concentration and purity of the DNA were detected by the electrophoresis gel imaging method, and the concentration of the sample to be tested was standardized to 10ng / ul. figure 1 shown;

[0036] The normal control DNA is considered qualified when it meets the following conditions: OD value A260 / A280 is between 1.8-2.0; the electrophoresis band is clear; there is no mutation in exon 7 of the PAH gene identified by sequencing, and the sequencing results are as follows: figure 2 shown.

[0037] 2. According to the conserved region of the PAH gene, the online software Primer 3 was used to design the HRM primer of exon 7 of the PAH gene. The size of the primer was determined to be 20bp, and the length of the PCR product was 204bp (synthesized by Sangon Bioengineering (Shanghai) C...

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Abstract

The invention discloses a method for detecting PAH (Polycyclic Aromatic Hydrocarbon) gene mutation by utilizing a high-resolution melting curve analysis technique. The method is characterized by comprising the following steps: adopting a silica gel adsorption method to extract the genome DNA of an oral epithelial cell/peripheral blood cell sample of a detected person, designing the third, sixth, seventh, eleventh and twelfth exon sequences of the PAH gene as detection primers of a template, and carrying out mutation detection on the gene sequences after PCR (Polymerase Chain Reaction) amplification according to the high-resolution melting curve analysis technique. The method has the advantages of high sensitivity, good specificity and high detection speed and can quickly screen common mutation sites of phenylalanine hydroxylase genes. The invention provides a new basis for the method for detecting PAH gene mutation.

Description

technical field [0001] The invention relates to a detection method for phenylalanine hydroxylase gene mutation, which belongs to the technical field of molecular biology. Background technique [0002] The phenylalanine hydroxylase (PAH) gene is located on chromosome 12q23.2, with a total length of about 90kb, including 13 exons and 12 introns. The length of the exon is a single strand of 1353bp, the mRNA is translated into an enzyme monomer containing 451 amino acids, and the monomer is polymerized into a functional PAH enzyme. PAH gene mutations can lead to liver phenylalanine hydroxylase deficiency, and phenylalanine cannot be converted to tyrosine normally, resulting in metabolic disorders in the liver, leading to phenylketonuria (PKU) in children. Phenylketonuria is an autosomal recessive genetic disease with abnormal amino acid metabolism. The incidence rate in Europe and the United States is 1 / 10000, in my country it is 1 / 16000, and the heterozygote frequency is 1 / 50...

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/686C12Q2527/107C12Q2563/107C12Q2600/156
Inventor 张奕傅咏南王校毛丹丹卞雪莲
Owner 上海中优医药高科技股份有限公司
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