Primer, kit and analysis method for ADTKD gene mutation detection

A kit and primer sequence technology, applied in the field of ADTKD gene mutation detection, can solve problems such as no genes involved, and achieve the effects of saving detection costs, wide application models, accurate genotyping and mutation site information

Pending Publication Date: 2021-09-17
SHANGHAI JIAO TONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] US20210169827A1 relates to compositions and methods for the diagnosis and treatment or prevention of proteinopathies, in particular MUC1-associated renal disease (ADTKD-MUC1 or MKD), retinitis pigmentosa (e.g. due to rhodopsin mutations), caused by Umod mutations ( ADTKD-Umod) caused autosomal dominant tubular-interstitial nephropathy, the patent disclosed only involves the detection of UMOD and MUC1 in the five pathogenic genes, and does not involve the simultaneous detection of genes HNF1B, REN, SEC61A1

Method used

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  • Primer, kit and analysis method for ADTKD gene mutation detection
  • Primer, kit and analysis method for ADTKD gene mutation detection
  • Primer, kit and analysis method for ADTKD gene mutation detection

Examples

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Embodiment 1

[0129] This embodiment provides a genetic diagnosis and genotyping method for ADTKD, covering UMOD, MUC1, HNF1B, REN, SEC61A1 five genes currently reported in the literature for all mutation sites that affect the coding protein region mutation diagnosis, including 14 Mutation analysis methods for PCR amplification primers, library construction kits, 16 sample mixed samples on-machine sequencing index adapter sequences and long-fragment sequencing sequences.

[0130] In this embodiment, the DNA amplification enzyme is selected from Takara's DNA polymerase;

[0131] The magnetic beads for purification are selected from AMPure XP Beads of Beckman Company.

[0132] Wherein, the preparation method of the magnetic beads for purification is:

[0133]1) Resuspend AMpure XP beads, take out 500ul, centrifuge at 16000rpm for 1min, put it on a magnetic stand, take out the supernatant and store it in a new tube.

[0134] 2) Add 1ml of water to the magnetic beads, resuspend, centrifuge at...

Embodiment 2

[0150] Based on the genetic diagnosis and genotyping kit for detecting ADTKD provided in Example 1, this example establishes an amplification system.

[0151] Specifically, when using this kit to detect MUC1 gene mutations, the PCR reaction mixture system is: 50 μl of each reaction mixture system, containing 200 ng of genomic DNA template, 10 μl of 5×Reaction Buffer, 4 μl of dNTP (2.5mMeach), DNA Polymerase 1 μl, Primer F (10 μM primer, HPLC grade) 1 μl, Primer R (10 μM primer, HPLC grade) 1 μl, add ddH 2 O to a total volume of 50 μl. Primer F and primer R are selected from M27-28, and M27-M28 corresponds to the MUC1 gene.

[0152] When the MUC1 gene PCR reaction system is established, 8 tubes can be amplified at the same time.

[0153] The thermal cycle conditions of the PCR reaction are as follows: preheat the hot lid at 105°C, heat at 98°C for 5 minutes, and then enter 30 cycles (using two-step thermal cycle conditions): denaturation temperature at 98°C for 10s (1st to 30...

Embodiment 3

[0182] Due to the different amplification efficiencies of PCR reactions, it is necessary to optimize the mixing ratio of PCR products in order to obtain results with consistent sequencing abundance. This example provides how to obtain the mixed sample volume for each PCR product.

[0183] This example provides a mixing method for obtaining consistent sequencing abundance of 13 PCR products. By adjusting the ratio of 13 PCR product mixtures, the effects of amplification efficiency and recovery efficiency during library construction were adjusted. By mixing and loading samples in equal volumes, the proportion of the final number of sequencing reads is adjusted to adjust the mixing ratio, so as to realize the output of sequencing data in equal proportions for multiple targeted bands.

[0184] The 13 amplified regions refer to the amplified regions corresponding to the 13 pairs of primers. Among them, M1-M6, M23-M24 correspond to UMOD gene, M7-M10 correspond to SEC61A1 gene, M11...

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Abstract

The invention relates to a primer, a kit and an analysis method for ADTKD gene mutation detection. The technical scheme provided by the invention comprises the steps of PCR primer design, replacement magnetic bead, enzyme and applicable system design and the like. Through optimized screening of a series of reagents, gene mutation detection of different samples, different types and the like is realized. According to the method, gene segments of five genes are targeted, the sequencing length ranges from 1.6 kbp to 3 kbp, the genes do not need to reach a short segment of about 300 bp, a targeted full-length sequence is directly obtained, and meanwhile through cyclic consistent sequence analysis, the detection accuracy of mutation and indel is higher. Especially for the MUC1 gene, the method overcomes the defect that the WES cannot accurately splice and compare the repetitive regions of the gene, can obtain an accurate circulating consistent sequence, visually identifies the number of the repetitive regions and mutation sites, and solves the problem that the WES cannot detect the mutation of the gene.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a primer, a kit and an analysis method for ADTKD gene mutation detection. Background technique [0002] Autosomal dominant tubulointerstitial kidney disease (autosomaldominant tubulointerstitial kidney disease, ADTKD) is a type of interstitial kidney disease with genetic predisposition and familial clustering. Patients with this type of renal disease eventually enter end-stage renal disease (ESRD for short), which brings a heavy burden to individuals, families and society. [0003] In 2015, the Global Kidney Outcomes Organization (KDIGO for short) reached a consensus on the renaming, clinical manifestations, renal pathology, diagnosis and treatment of autosomal dominant tubulointerstitial nephropathy. So far, 5 ADTKD pathogenic genes (UMOD, MUC1, HNF1B, REN, SEC61A1) have been found and corresponding genotypes have been identified. Because the B-ultrasound of AD...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2535/122C12Q2537/165
Inventor 赵明珠李红梅蒋更如林芙君
Owner SHANGHAI JIAO TONG UNIV
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