Glu504lys detection genotyping kit based on AllGlo probe and genotyping method thereof

A kit and probe technology, applied in the field of single nucleotide polymorphism, can solve the problems of ALDH2 activity change, loss of drug effect, nitroglycerin can not produce nitric oxide, etc., and achieve strong fluorescence signal, simple process, and detection. Inexpensive effect

Inactive Publication Date: 2016-09-28
ZHONGSHAN HOSPITAL XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is a SNP site in the 12th exon of ALDH2—Glu504lys. This SNP site will lead to changes in the activity of ALDH2, which will cause nitroglycerin to fail to produce nitric oxide and lose its efficacy

Method used

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  • Glu504lys detection genotyping kit based on AllGlo probe and genotyping method thereof
  • Glu504lys detection genotyping kit based on AllGlo probe and genotyping method thereof
  • Glu504lys detection genotyping kit based on AllGlo probe and genotyping method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0038] The Glu504lys detection and typing kit based on the AllGlo probe of the present invention comprises the following components:

[0039] Real-time fluorescence quantitative PCR reagents, positive control and negative control.

[0040] ①Reagents for real-time fluorescence quantitative PCR include the following components: 10×Taq buffer (10×Taq buffer includes 100mmol Tris-HCl and 500mmol KCl), 10mmol MgCl 2 , 5u / μL Taq Hotstart DNA polymerase, 10m moldNTPs mixture, 50×Low ROX, 100mL nuclease-free water, 10μmol / L Glu504lys specific forward primer, 10μmol / L Glu504lys specific reverse primer and AllGlo probe .

[0041] ② Positive controls include: positive homozygous control substance 1, positive homozygous control substance 2, and positive heterozygous control substance. The positive homozygous control substance 1 is a DNA sample whose Glu504lys type is AA, the positive homozygous control substance 2 is a DNA sample whose Glu504lys type is GG, and the positive heterozygous...

Embodiment 2

[0044] The detection and typing of Glu504lys in peripheral blood includes the following steps:

[0045] 1. Collect EDTA anticoagulated peripheral blood:

[0046] Take 2 mL of fresh blood samples and put them into EDTA anticoagulant tubes and divide them into multiple tubes. Each tube is divided into 400-500 μL, and 200 μL is used for DNA extraction. The rest of the whole blood samples are stored at -80°C.

[0047] 2. Extract DNA:

[0048] Use Genomic Blood DNA Extraction Kit (DP348) produced by Tiangen Biotechnology Co., Ltd. to extract DNA from the sample (whole blood) according to the operating instructions, 200μL of LEDTA anticoagulated whole blood according to the instructions, and finally use 50μL of elution buffer TB dissolves the DNA, and measures the concentration and purity on the nucleic acid spectrophotometer Nano Drop2000. The purity A260 / A280 ratio is between 1.7 and 1.9, and the extracted DNA with a concentration of 10-50 ng / μL is used for SNP typing detection. ...

Embodiment 3

[0063] Example 3. SNP detection and typing of tissue samples

[0064] 1. Tissue sample processing:

[0065] Tissues (the amount of spleen tissue should be less than 10 mg) should be crushed and processed into a cell suspension, then centrifuged at 10,000 rpm (~11,200×g) for 1 min, drain the supernatant, add 200 μL buffer GA (Tiangen DNA Extraction Kit DP304), and shake until completely suspended;

[0066] 2. DNA extraction and enrichment of tissue samples:

[0067] Use the DNA extraction kit (DP304) produced by Tiangen Biotechnology Co., Ltd. to extract DNA from tissue samples according to the operating instructions, and finally dissolve the DNA with 50 μL of elution buffer TE, and measure the concentration on the nucleic acid spectrophotometer NanoDrop2000. and purity;

[0068] 3. SNP detection and typing of tissue samples.

[0069] For subsequent steps, refer to steps 3-4 of Example 2.

[0070] Applicable range:

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Abstract

The invention relates to single nucleotide polymorphism, in particular to a Glu504lys detection genotyping kit based on an AllGlo probe and a genotyping method thereof. The kit comprises a real-time fluorescence quantification PCR (polymerase chain reaction) reagent, positive control and negative control, Glu504lys is an SNP locus in an ALDH2 gene of human chromosome 12 provided by NCBI. The genotyping method comprises the steps that firstly, DNA in an EDTA anti-freezing whole blood sample is extracted through a conventional method; secondly, the real-time fluorescence quantification PCR reagent provided by the Glu504lys detection genotyping kit based on the probe AllGlo is used for conducting real-time fluorescence quantification PCR amplification on the DNA; and thirdly, genotyping is conducted on the Glu504lys locus of the human gene ALDH2 according to detected fluorescence signals. On the premise of keeping high specificity and sensitivity of the probe AllGlo, the detecting price is lower than that of a direct sequencing method, the process is simpler, and consumed time is shorter.

Description

technical field [0001] The present invention relates to a single nucleotide polymorphism (SNP), in particular to an AllGlo probe-based Glu504lys detection and typing kit and typing method thereof. Background technique [0002] Single nucleotide polymorphism (single nucleotide polymorphism, SNP), mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is the most common type of heritable variation in humans. SNPs widely exist in the human genome, with an average of 1 in every 500-1000 base pairs, and it is estimated that the total number can reach 3 million or more. As a third-generation genetic marker, SNP is closely related to many phenotypic differences, drug susceptibility and disease susceptibility. The large number of SNPs in the genome makes it a powerful tool and plays a very important role in disease localization and cloning, drug design and testing, and basic biological research. [0003] Individualize...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 方宜臻林华月张忠英
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV
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