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Kit for screening of primary open-angle glaucoma

A kit and reagent technology, applied in the field of SNP, can solve the problem of unseen association between glaucoma and RAMP2 gene mutation site

Active Publication Date: 2019-04-30
SICHUAN PROVINCIAL PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, there is no association between glaucoma and the mutation site of the RAMP2 gene

Method used

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  • Kit for screening of primary open-angle glaucoma
  • Kit for screening of primary open-angle glaucoma
  • Kit for screening of primary open-angle glaucoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0032] Embodiment Kit and method of use of the present invention

[0033] All components, content and method of use in the kit of the present invention are as follows:

[0034] 1. PCR amplification reagent (50 people):

[0035] The PCR amplification reagent is used to amplify a DNA sequence where the SNP site is located, and its composition is shown in Table 1.

[0036] Table 1 PCR amplification reagents

[0037] The PCR mixture in Table 1 includes Taq enzymes, dNTPs, magnesium ions and other components required for conventional PCR;

[0038]

[0039] The primer pair information is shown in Table 2.

[0040] Table 2 Primers used in RAMP2 gene amplification

[0041]

[0042] Attachment: Primer-1F / R primer amplification: Glu39Asp (c.117G>C), Glu54Lys (c.160G>A)

[0043]Primer-2F / R primer amplification: Phe103Ser(c.308T>C), Asn113Lysfs*10(c.338_339insA), Glu143Lys (c.427G>A), Ser171Arg(c.511A>C)

[0044] 2. RAMP2 gene variation typing detection reagent (50 people):

...

experiment example 1

[0076] Screening of experimental example 1 mutation site

[0077] 1. Method

[0078] DNA was extracted from 4763 POAG patients and 10953 healthy controls. Through next-generation sequencing technology, using Illumina TruSeq enrichment capture, HiSeq 2000 / 2500 sequence generator targeted screening of POAG candidate genes, so that the genomic DNA of each individual is enriched in the library. Libraries were sequenced using an automated Illumina HiSeq 2000 / 2500 sequencer. The sequence results were aligned with the sequence of UCSC hg19 (http: / / genome.ucsc.edu / ) using BWA-MEM (https: / / github.com / lh3 / bwa). SNPs and Indels were detected by using two databases, Samtools (http: / / samtools.sourceforge.net / ) and freebayes (https: / / github.com / ekg / freebayes). Based on the information from the RefSeq database, we used the Annovar (http: / / annovar.openbioinformatics.org / en / latest / ) database to classify SNVs into different functional categories based on their genetic location and expected i...

experiment example 2

[0083] Experimental example 2 The specificity test of the kit of the present invention for RAMP2 genotyping detection

[0084] 1. Method

[0085] The mutation sample DNA involved in Table 2 in Experimental Example 1 and the DNA of 20 healthy subjects were used as experimental materials, and the kits in the examples were used to perform detection according to the above-mentioned experimental method.

[0086] 1) DNA extraction

[0087] Take 2ml of whole blood (EDTA anticoagulated) from the patient, and extract its genomic DNA.

[0088] 2) Amplify the DNA fragment containing the detected SNP site by PCR, and the PCR amplification system of each mutation site is as follows:

[0089]

[0090]

[0091] Reaction conditions:

[0092]

[0093] PCR product detection:

[0094] Use 2% agarose gel electrophoresis to detect the PCR product, observe the effect of the PCR reaction, and determine the amount of it added as a template in subsequent reactions.

[0095] 3) Sanpshot t...

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Abstract

The invention discloses a kit for the screening of primary open-angle glaucoma. The invention provides an application of related reagents for detecting the human RAMP2 gene mutant sites: c.117G>C, c.160G>A, c.308T>C, c.338_339insA, c.427G>A and / or c.511A>C in the preparation of reagents for screening primary open-angle glaucoma. The invention also provides a glaucoma screening kit, which comprisesrelated reagents for detecting human RAMP2 gene mutant sites: c.117G>C, c.160G>A, c.308T>C, c.338_339insA, c.427G>A and / or c.511A>C.

Description

technical field [0001] The invention relates to the field of SNPs, in particular to SNPs related to glaucoma. Background technique [0002] Glaucoma is a common and irreversible severe blinding eye disease in the world. It is mainly characterized by optic nerve damage, manifested as optic ganglion cell apoptosis. Primary open-angle glaucoma (POAG) is the most common type of glaucoma, accounting for about 60% to 70% of all glaucoma; it can be divided into high intraocular tension glaucoma (HTG) and normal intraocular tension glaucoma ( NPG). Both genetic and environmental factors can cause POAG. From a genetic point of view, both single-gene mutations and polygenic mutations can cause POAG. Single genes responsible for POAG include MYOC (encoding myofibrin), OPTN (encoding optic neurin), WDR36 (encoding WD overlapping domain 36), TBK1 (encoding TANK-binding kinase 1), and ASB10 (encoding ankyrin repeat and SOCS box 10). Multiple gene mutations lead to POAG genes includin...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 杨正林龚波张侯斌黄璐琳林婴
Owner SICHUAN PROVINCIAL PEOPLES HOSPITAL
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