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PCR primer set and amplification system for amplifying exons 1-33 of human PKD1 gene

An amplification system and exon technology, applied in the field of genetic disease gene detection, can solve the problems of pseudogene interference, poor timeliness, complicated operation process, etc.

Pending Publication Date: 2019-07-05
CARRIER GENE TECH SUZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the problems of pseudogene interference, high cost, complicated operation process and poor timeliness in the prior art, the present invention discloses a simple and efficient method for detecting PKD1 gene mutation

Method used

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  • PCR primer set and amplification system for amplifying exons 1-33 of human PKD1 gene
  • PCR primer set and amplification system for amplifying exons 1-33 of human PKD1 gene
  • PCR primer set and amplification system for amplifying exons 1-33 of human PKD1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 (this embodiment is suitable for amplifying PKD1 gene exon 2-5a, exon 5b-6, exon 7-8, exon 11, exon 13-15a, exon 15b , exon 15c, exon 15d-16, exon 17-20, exon 21, exon 22, exon 23-24, exon 25-26, exon 27-30 , exons 31-33)

[0031] 1. The samples and reagents that need to be prepared are:

[0032] Genomic DNA (gDNA) extracted from the sample to be tested, with A260-280 between 1.8-2.0, diluted to 10ng / μL with nuclease freewater; primers with the base sequence shown in SEQ ID NO:1-36, with nuclease free water Dilute to 10 μmmol / L; RNase enzyme diluted to 5mU / μL; 5U / μL TaKaRa LA Taq DNA polymerase; 2.5mmol / LdNTP mixture; TaKaRa 2×GC Buffer Ⅰ.

[0033] 2. Use the gDNA of the sample to be tested as a template to amplify exons 1-33 of the PKD1 gene. Prepare a PCR reaction system in a sterilized PCR tube according to the following system.

[0034] The 20μL PCR reaction system is as follows:

[0035] PCR components Added volume (μL) gDNA (10ng...

Embodiment 2

[0044] Example 2 (this example is suitable for amplifying PKD1 gene exon 1, exon 9-10, and exon 12) 1. The samples and reagents to be prepared are:

[0045] Genomic DNA (gDNA) extracted from the sample to be tested, with A260-280 between 1.8-2.0, diluted to 10ng / μL with nuclease freewater; primers with the base sequence shown in SEQ ID NO:1-36, with nuclease free water Dilute to 10μmmol / L; Dilute RNase to 5mU / μL; 5U / μL TaKaRa LA Taq DNA polymerase; 2.5mmol / LdNTP mixture; TaKaRa 2×GC Buffer Ⅱ.

[0046] 2. Use the gDNA of the sample to be tested as a template to amplify exons 1-33 of the PKD1 gene. Prepare a PCR reaction system in a sterilized PCR tube according to the following system.

[0047] The 20μL PCR reaction system is as follows:

[0048] PCR components Added volume (μL) gDNA (10ng / μL) 1 2×GC BufferⅡ 10 2.5mmol / L dNTP mix 3.2 Forward primer (10μmmol / L) 0.4 Reverse primer (10μmmol / L) 0.4 RNase (5mU / μL) 2 TaKaRa LA Ta...

Embodiment 3

[0053] Example 3 (This example uses nested PCR and the amplification system of the present invention to verify the NGS data respectively)

[0054] 1. This example involves a family with polycystic kidney disease, including the proband, the younger brother of the proband, the father of the proband and the mother of the proband, a total of four members. Among them, the clinical symptoms of the proband were polycystic kidney disease with ejaculatory duct cyst and azoospermia; the clinical symptoms of the proband’s younger brother were polycystic kidney disease, the sperm motility and density were normal, and the sperm deformity rate was 99%; the father had polycystic kidney disease, and the mother The phenotype was normal. Through the bioinformatics analysis of the second-generation high-throughput data of the whole exon of the four samples of the family and one control sample and the genetic counseling analysis according to the "ACMG Genetic Variation Classification Standards an...

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Abstract

The present invention discloses a PCR primer set and an amplification system for amplifying exons 1-33 of a human PKD1 gene (Gene ID:5310), comprises a primer set capable of distinguishing the PKD1 gene and a pseudo gene thereof, and a required PCR reaction buffer using the primer set to amplify the exons 1-33 of the PKD1 gene. Compared with traditional nested PCR and long fragment PCR amplified PKD1 gene, the PCR primer set and amplification system have advantages of being convenient for operation, rapid, high in efficiency, etc., can be used for direct detection of adult type polycystic kidney disease-causing gene PKD1 gene mutations, and can also be used for verification of the PKD1 gene mutations in next-generation sequencing results.

Description

technical field [0001] The invention relates to the field of gene detection for genetic diseases, and more specifically relates to a primer set and an amplification system capable of distinguishing the adult polycystic kidney disease treatment gene PKD1 gene and its pseudogene. Background technique [0002] Autosomal dominant polycystic kidney disease (ADPKD) is usually a late-onset multisystem disease with an incidence of 1 / 400-1 / 1000. Patients often have vesicles of different sizes in their bilateral kidneys when they are 30-50 years old. Characterized by: Bilateral renal cysts with involvement of other organs including liver, seminal vesicles, pancreas, and arachnoid; vascular abnormalities including intracranial aneurysms, aortic root dilatation, and abnormalities of the thoracic aorta; mitral valve Prolapse and abdominal wall hernia. Renal manifestations include hypertension, renal pain, and renal insufficiency. Approximately 50% of ADPKD patients develop end-stage re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 汪进平罗俊峰陈云弟
Owner CARRIER GENE TECH SUZHOU CO LTD
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