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Kit for detecting mutation of pathogenic genes of phenylketonuria

A phenylketonuria and kit technology, which is applied in the field of kits for detecting mutations in the pathogenic gene of phenylketonuria, can solve the problems of cumbersome, powerless prenatal diagnosis, and long time-consuming biochemical detection, and reduce the incidence rate Effect

Inactive Publication Date: 2017-12-29
中科基因生物科技(江苏)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Practice has shown that the biochemical detection method is time-consuming and cumbersome, and is only applicable to babies who have already been born, and is powerless for prenatal diagnosis

Method used

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  • Kit for detecting mutation of pathogenic genes of phenylketonuria
  • Kit for detecting mutation of pathogenic genes of phenylketonuria
  • Kit for detecting mutation of pathogenic genes of phenylketonuria

Examples

Experimental program
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Effect test

Embodiment 1

[0046] A kit for detecting mutations in the phenylketonuria pathogenic gene PAH for one person, including the following components: (1) 13 pairs of primers for PCR amplification and sequencing of exons 1 to 13 of the PAH gene, each primer 1OD, primer See Table 1 for the sequence;

[0047] (2) PCR amplification reagent: 7.5 μL of Taq enzyme mixture (dNTP, 10×PCR reaction buffer, MgCl 2 );

[0048] (3) PCR product purification reagent: 1.6 μL SAP enzyme mixture (SAP enzyme, ExoI enzyme, deionized water);

[0049] (3) Sequencing reagents: 2.2 μL BigDye mix (Bigdye, 5×seq), 2.5 μL EDTA, 30 μL ethanol solution (100%), 100 μL ethanol solution (70%), 8 μL Hi-Di.

[0050] This kit is stored at -20°C, and repeated freezing and thawing should be avoided as much as possible.

Embodiment 2

[0052] The steps for using a kit for detecting whether a mutation occurs in the phenylketonuria pathogenic gene PAH for one person include:

[0053] (1) extract the genomic DNA of the sample;

[0054] (2) PAH gene PCR amplification

[0055] For the exon 1-13 region of PAH gene, primers were designed according to Table 2 for PCR amplification. Each reaction system has a total volume of 16u 1, including 7.5 μL of Taq enzyme mixture (dNTP, 10×PCR reaction buffer, MgCl 2 ), deionized water 5.5 μL, primer pair (10uM) 1 μL, genomic DNA (100ng / ul) 1ul. The reaction conditions are 95°C for 15 minutes, 14 cycles (-0.5°C / cycle) of 94°C for 30 seconds, 63°C for 30 seconds, and 72°C for 45 seconds; then 30 cycles of 94°C for 30 seconds and 56°C for 30 seconds , 72°C for 45 seconds, and finally 72°C for 10 minutes.

[0056] (3) Purification of PCR products

[0057] Each reaction has a total volume of 5.6 μL. 4 μL of PCR product was added, and then 1.6 μL of SAP enzyme mixture (SAP en...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a kit for detecting whether pathogenic genes of phenylketonuria are mutated. The kit contains the following main components: (1) 13 pairs of PCR (Polymerase Chain Reaction) amplification and sequencing primers of 1-13 exon areas of PAH (Polycyclic Aromatic Hydrocarbon) genes; (2) PCR amplification reagent; (3) PCR product purifying reagent; and (4) DNA sequencing reagent. The kit is used for detecting mutants of the PAH genes of the pathogenic genes of the phenylketonuria, and has the advantages that carriers and patients with the pathogenic genes can be rapidly and conveniently checked, the benefit for prenatal diagnosis and early stoppage of birthing of child patients is achieved, and the morbidity of the phenylketonuria is reduced.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a kit for detecting whether a mutation occurs in a pathogenic gene of phenylketonuria. Background technique [0002] The prior art discloses that phenylketonuria (Phenylketonuria, PKU) is a genetic metabolic disease, which was discovered by Dr. Folling of Norway in 1934. It is named after a large amount of phenylpyruvate in the patient's urine. Studies have shown that the disease is due to the decrease in the activity of phenylalanine hydroxylase (PAH) in the body or the lack of its coenzyme tetrahydrobiotine, which leads to the obstruction of the metabolism of phenylalanine to tyrosine, and the concentration of phenylalanine in blood and tissues Increased, urine phenylpyruvate, phenylacetic acid and phenyllactic acid significantly increased, known as "phenylketonuria". The survey shows that although the disease is a genetic metabolic disease, it is not uncommon....

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6883C12Q1/6869C12Q2600/156C12Q2600/16C12Q2537/143C12Q2531/113
Inventor 向天敏徐沁梅艳巧景丹丹
Owner 中科基因生物科技(江苏)有限公司
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