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PKD1 gene mutation detection kit and detection method

A kit and gene technology, applied in the field of medical molecular biology detection, can solve the problems of long time, unsatisfactory primer stability, and inability to effectively eliminate pseudogene interference.

Active Publication Date: 2015-04-22
北京圣谷智汇医学检验所有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the applicant found that in the method disclosed in this article, the interference of pseudogenes cannot be effectively eliminated after the amplification of long fragments of some primers, and the stability of the primers is still not ideal, and it can only be completed after three times of PCR reaction conditions. Amplification of the entire gene takes longer and is less efficient
This method uses the Illumina Miseq sequencer for detection, which takes 5 to 10 days to complete the sequencing, and the efficiency is low
Moreover, because the GC content of exon 1 sequence of PKD1 gene is greater than 85%, the quality of exon 1 sequencing is poor

Method used

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  • PKD1 gene mutation detection kit and detection method
  • PKD1 gene mutation detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1: Using the true gene-specific primers (E2, T3, K3, R5, K5) of the PKD1 gene exon 2-46 region to perform long-fragment PCR and high-throughput sequencing to detect PKD1 gene mutations

[0087] On the premise of obtaining the informed consent of the subjects, the PKD1 gene mutation was detected by long-fragment PCR combined with high-throughput sequencing for 70 subjects.

[0088] Use the OMEGA Genomic DNA Extraction Kit (purchased from OMEGA, USA) to extract the genomic DNA from the peripheral blood of the subject, and use a spectrophotometer or other testing instruments to detect the DNA concentration and purity of the extracted DNA. The DNA concentration is greater than 50 ng / μl, and the volume is greater than 30μl, A260 / A280 between 1.6-2.0, as a DNA template. The DNA templates from each subject were equally divided into five separate reaction tubes, and long-segment PCR primers E2, T3, K3, R5, and K5 (sequences are shown in Table 1) were added to each of th...

Embodiment 2

[0141] Embodiment 2: The primer specificity comparison of the method of PKD1 gene-specific T3 primer (SEQ ID NO:3 and 4) and R5 primer (SEQ ID NO:7 and 8) and Adrian Y.Tan etc.

[0142] On the premise of obtaining the informed consent of the subject, peripheral blood was collected from one subject, and the genomic DNA of the subject's peripheral blood was extracted with the OMEGA Genomic DNA Extraction Kit (purchased from OMEGA Company, USA). Spectrophotometer or other detection equipment to detect DNA concentration and purity, DNA concentration greater than 50ng / μl, volume greater than 30μl, A260 / A280 between 1.6-2.0, used as template DNA.

[0143] Using primers PKD1_NGS_2-12F, PKD1_NGS_2-12R, PKD1_NGS_13-21F, PKD1_NGS_13-21R, PKD1_NGS_22-34F, PKD1_NGS_22-34R and its PCR reaction system and PCR reaction conditions to amplify exon 2-34 according to the method of Adrian Y.Tan et al. In addition, E2 primers (SEQ ID NOs: 1 and 2), T3 primers (SEQ ID NOs: 3 and 4), K3 primers (SEQ...

Embodiment 3

[0150] Embodiment 3: The primer stability comparison of E2, T2, R5 primer and the method such as Adrian Y.Tan

[0151] On the premise of obtaining the informed consent of the subjects, peripheral blood was collected from 8 subjects, and the genomic DNA of the subjects' peripheral blood was extracted with the OMEGA Genomic DNA Extraction Kit (purchased from OMEGA Company, USA). Spectrophotometer or other detection equipment to detect DNA concentration and purity, DNA concentration greater than 50ng / μl, volume greater than 30μl, A260 / A280 between 1.6-2.0, used as template DNA.

[0152] The primers PKD1_NGS_2-12F, PKD1_NGS_2-12R of the method such as Adrian Y.Tan, PKD1_NGS_2-12R and its PCR reaction system and PCR reaction conditions, adopt the E2 primer (SEQ ID NO:1 and 2) primers and T3 primers (SEQ ID NO:3 and 4) primers and the reaction system described in the above table 2 and the PCR reaction conditions described in the above table 3 for amplifying No. 8-12 exon regions, re...

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Abstract

The invention provides a primer set, a kit and a detection reaction system for detecting PKD1 gene mutation through the long fragment PCR and high-throughput sequencing technology, a non-diagnosis-purpose method for external PKD1 gene mutation detection, a non-diagnosis-purpose method for external PKD1 gene analysis and a method for detecting new mutation sites on the PKD1 gene. According to the method, the primer set is used for carrying out long fragment PCR amplification on the PKD1 gene of a sample, and detecting or analyzing is carried out through high-throughput sequencing. The autosomal dominant genetic polycystic nephrosis (adult polycystic nephrosis) can be diagnosed through the assistance of a detection result, previous unknown new mutation on a plurality of PKD1 real genes can be obtained and supplied to doctors or researchers so that the relevance between the mutation and the adult polycystic nephrosis can be studied.

Description

technical field [0001] The invention relates to the technical field of medical molecular biology detection, in particular to a primer set, a kit for detecting PKD1 gene mutations related to hereditary polycystic kidney disease, and a method for detecting PKD1 gene mutations using the primer set and kit. Background technique [0002] 1. Disease and genetic background [0003] Autosomal dominant polycystic kidney disease (adult polycystic kidney disease, APKD, also known as adult polycystic kidney disease) is a common congenital disease. Age-onset is a common hereditary multiple cystic kidney disease with an incidence of about 1 / 1,000-1 / 400, accounting for about 5% of end-stage renal disease. The disease slowly forms cysts and enlargements in the kidneys in middle age, eventually leading to kidney failure. The diagnosis of autosomal dominant polycystic kidney disease mainly depends on imaging or molecular genetic testing. For imaging detection, the sensitivity of detection ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G06F19/22
CPCC12Q1/6869C12Q1/6883C12Q2600/156G16B30/00C12Q2535/122
Inventor 杨贵江张泽樘赵雪燕杨扬焦慧
Owner 北京圣谷智汇医学检验所有限公司
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