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Primer, kit and method for detecting PAH gene mutation

A kit, the technology of PAH-6F, which is applied in the fields of life science and biology, can solve the problems of unclear mechanism affecting RNA splicing and PAH activity, so as to reduce cost and difficulty, reduce the amount of samples and reagents used, and reduce detection cost effect

Pending Publication Date: 2020-04-24
北京艾迪康医学检验实验室有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ellingsen et al. have confirmed that the 96A>G mutation produces a new splicing site, which affects RNA splicing, but the mechanism of the effect of the R243Q mutation on PAH activity is still unclear

Method used

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  • Primer, kit and method for detecting PAH gene mutation
  • Primer, kit and method for detecting PAH gene mutation
  • Primer, kit and method for detecting PAH gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Primers for detecting mutations in exons 6 and 7 of the PAH gene, which are amplification primers specifically designed for exons 6 and 7 of the PAH gene:

[0040] A kit for detecting mutations in exons 6 and 7 of the PAH gene, including

[0041] (i) blood / tissue DNA extraction reagents;

[0042] (ii) detection system PCR reaction solution;

[0043] (iii) Sequencing system reagents;

[0044] (iv) Positive control substance and negative control substance.

[0045] Among them, blood / tissue DNA extraction reagents can be purchased from commercial reagents such as Tiangen DNA Extraction Kit.

[0046] Detection system PCR amplification reaction solution includes: 2×PCR Buffer; 2mM dNTPs; KOD FX DNA Polymerase (1U / μl); PAH-6F (10μM), PAH-6R (10μM), PAH-7F (10μM), PAH-7R (10 μM).

[0047] PAH-6F:TGTAAAACGACGGCCAGTCCCGACTCCCTCTGCTAA

[0048] PAH-6R:AACAGCTATGACCATGGCCTCAATCCTCCCCCAA

[0049] PAH-7F: TGTAAAACGACGGCCAGTCTATGTCCCTGGGCAGTTATG

[0050]PAH-7R:AACAGCTATGACCATG...

Embodiment 2

[0055] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Biology):

[0056] (1) Extract tissue DNA from blood:

[0057] 1) Take 300 μl of blood and add 900 μl of erythrocyte lysate, mix by inversion, and let stand at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 12,000rpm for 1min, suck off the supernatant, leave the white blood cell pellet, add 200μl buffer GA, shake until thoroughly mixed.

[0058] 2) Add 20 μl proteinase K solution and mix well.

[0059] 3) Add 200 μl of buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0060] 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0061] 5) Add the solution and floccu...

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Abstract

The invention discloses primers for detecting hotspot mutation of a phenylketonuria related gene PAH gene. The primers comprise primers for amplifying the sixth exon and the seventh exon of the PAH gene; by adopting a Sanger sequencing technology, the primers can be used for rapidly detecting mutation of the sixth exon and the seventh exon of the PAH gene in the body of a patient with phenylketonuria. The detection result completed by the kit is accurate, and the kit has important reference significance for rapid diagnosis of phenylketonuria patients.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and in particular relates to a primer, a method and a kit for detecting mutations of exons 6 and 7 of PAH gene. Background technique [0002] Phenylketonuria (Phenylketonuria'PKU) is an autosomal recessive genetic disease caused by the lack or activity of phenylalanine hydroxylase (Phenylalaninehydroxylase, PAH) in the liver. At present, there are 564 kinds of mutations reported in the world, and more than 100 kinds of mutations have been reported in my country. The incidence of PKU in the Chinese population is 1 / 11000. Since 1998 in our province, a total of 1,606,213 newborns have been screened, and a total of 63 children have been diagnosed with PKU, with an incidence rate of 1 / 24,700. Children are usually normal at birth. Symptoms usually appear at 3-6 months. Symptoms are evident around 1 year old. Early vomiting, irritability, growth retardation and other phenomena. Untrea...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6869C12N15/11
CPCC12Q1/6883C12Q1/6869
Inventor 林筱剑
Owner 北京艾迪康医学检验实验室有限公司
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