Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Amplification and sequencing primers and kit for mutation detection of all coding regions of gene of androgen receptor

A technology for androgen receptor and sequencing primers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve the effect of reducing manpower and material resources, avoiding inconvenience and pollution, and avoiding difficulties in primer configuration

Active Publication Date: 2014-08-20
THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
View PDF4 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no comprehensive and accurate test kit for androgen receptor gene mutation sequencing, which can be used for clinical diagnosis

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Amplification and sequencing primers and kit for mutation detection of all coding regions of gene of androgen receptor
  • Amplification and sequencing primers and kit for mutation detection of all coding regions of gene of androgen receptor
  • Amplification and sequencing primers and kit for mutation detection of all coding regions of gene of androgen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] A set of specific primers for androgen receptor amplification and sequencing, including the amplification of the first exon and the sequencing primers are SEQ ID NO.1-SEQ ID NO.9, the amplification of the second exon And sequencing primers are SEQ ID NO.10 and SEQ ID NO.11, the amplification and sequencing primers of the third exon are SEQ ID NO.12 and SEQ ID NO.13, the amplification and sequencing of the fourth exon The primers are SEQ ID NO.14-SEQ ID NO.16, the amplification and sequencing primers of the fifth exon are SEQ ID NO.17 and SEQ ID NO.18, and the amplification and sequencing primers of the sixth exon are SEQ ID NO.19 and SEQ ID NO.20, the amplification and sequencing primers of the seventh exon are SEQ ID NO.21-SEQ ID NO.23, the amplification and sequencing primers of the eighth exon are SEQ ID NO.24 and SEQ ID NO.25.

Embodiment 2

[0046] A kit for amplifying and sequencing the androgen receptor, consisting of the following reagents:

[0047] Reagent A: Consists of 2mL buffer, containing 2×SYBR® Green I dye, 0.2mM dNTPs (including dATP, dCTP, dGTP, dTTP), 80mM Tris-HCl, 80mM KCl, 40mM (NH4)2SO4, 6mM MgSO4, 100U Taq DNA polymerase;

[0048] Reagent E1-A: 30 μL aqueous solution of sequencing primers targeting the first exon E1-A of AR, containing 4 μM SEQ ID NO.1;

[0049] Reagent E1-B: 30 μL aqueous solution of sequencing primers targeting the first exon E1-B of AR, containing 4 μM SEQ ID NO.3;

[0050] Reagent E1-C: 30 μL aqueous solution of sequencing primers targeting the first exon E1-C of AR, containing 4 μM SEQ ID NO.7;

[0051] Reagent E1-D: 30 μL aqueous solution of sequencing primers targeting the first exon E1-D of AR, containing 4 μM SEQ ID NO.8;

[0052] Reagent E2: 30 μL aqueous solution of sequencing primers targeting AR second exon E2, containing 4 μM SEQ ID NO.11;

[0053] Reagent E3: ...

Embodiment 3

[0072] Real-time fluorescent quantitative PCR (real-time FQ PCR) is used to amplify all exons of the androgen receptor with the kit of the present invention. First, calculate the required amount of each reagent according to the number of experimental specimens. The experimental steps are as follows:

[0073] 1. Amplification reaction solution configuration: Prepare the reaction solution (1 test sample) in order, add 120 μL of water, 150 μL of reagent A, and 30 μL of DNA sample, a total of 300 μL; mix well.

[0074] 2. Add 25μL amplification reaction solution to each tube sequentially, and cover the cap well.

[0075] 3. Amplify according to the PCR program designed for the experiment: pre-denaturation at 95°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 35 seconds. During the extension period, the fluorescence value was obtained for 35 cycles, and finally stored at 25°C; Remove in time for sequencing.

[0076]...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses amplification and sequencing primers and kit for the mutation detection of all coding regions of a gene of an androgen receptor The kit comprises a reaction solution A, reaction tubes and primers (E1-A, E1-B, E1-C, E1-D, E2, E3, E4, E5, E6, E7 and E8), wherein the reaction solution A is applied to PCR (Polymerase Chain Reaction) amplification, the reaction tubes are coated with the primers, and the primers are used for sequencing. According to the invention, all eight exons of a human androgen receptor can be amplified under the same reaction conditions once and then are subjected to sequencing by the sequencing primers; a PCR reaction system is provided with a fluorescent dye, so that amplification results can be learnt directly through the change of fluorescence values, and inconvenience and pollution resulting from electrophoresis are avoided; the primers are directly coated into the reaction tubes, so that the difficulty of primer allocation is avoided; the number of CAG replication in a first exon can be detected; the kit can be applied to the detection on clinical specimens, and more biological information can be provided.

Description

technical field [0001] The present invention relates to a molecular biology method based on polymerase chain reaction (PCR) and DNA sequencing technology, in particular to an amplification and sequencing primer and kit for detecting mutations in the full coding region of the androgen receptor (AR) gene . Background technique [0002] Androgen insensitivity syndrome (AIS) is a type of X-chromosome recessive genetic disease causing abnormal sex development (DSD), and its pathogenesis is closely related to AR gene mutation. The patient's karyotype is 46,XY, and the androgen receptor on the androgen target organ is defective, resulting in the loss of all or part of the normal effects of androgen. According to clinical manifestations, it can be divided into two subtypes: complete androgen insensitivity syndrome (CAIS) and incomplete androgen insensitivity syndrome (PAIS). [0003] The androgen receptor gene encoding the androgen receptor protein is located at Xq11-12, and the...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6869C12Q2531/113C12Q2563/107
Inventor 陈必成周蒙滔庄成乐洪炜龙徐慧英叶文乐王张阳朱哲慧潘晓东褚光宇
Owner THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com