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Method and primer pair for detecting CAG trinucleotide repetition sequence of HTT gene

A nucleotide sequence and trinucleotide technology, applied to a method for detecting the HTT gene CAG trinucleotide repeat sequence and the field of primer pairs, can solve the problem of difficulty in obtaining sequencing maps and high GC content of HTT gene CAG repeat fragments , clinical diagnosis of Huntington's disease and other problems

Pending Publication Date: 2019-08-30
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high GC content of the CAG repeat fragment of the HTT gene and the complex secondary structure, it is often difficult to obtain an ideal sequencing map, which has caused difficulties in the clinical diagnosis of Huntington's disease

Method used

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  • Method and primer pair for detecting CAG trinucleotide repetition sequence of HTT gene
  • Method and primer pair for detecting CAG trinucleotide repetition sequence of HTT gene
  • Method and primer pair for detecting CAG trinucleotide repetition sequence of HTT gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Design and synthesize primer sets, including the following steps:

[0051] Step 1.1: According to the HTT gene CAG trinucleotide repeat sequence and related literature, design specific amplification primers, and the forward primer is labeled with the fluorescent group FAM.

[0052] FAM is carboxyfluorescein, as a blue fluorescent labeling dye, synthesized by Huada Genomics Co., Ltd.

[0053] The designed primer sequences are:

[0054] HTT F: 5'-FAM-TGAAGGCCTTCGAGTCCCTCAAGTCCTTC-3'

[0055] HTT R: 5'-CGGCTGAGGAAGCTGAGGAG-3'

[0056] Wherein, F refers to the forward primer, and R refers to the reverse primer.

[0057] Step 1.2: Synthesize the primers designed in 1.1.

Embodiment 2

[0059] Genomic DNA is extracted from the sample to be tested, comprising the following steps:

[0060] Step 2.1: Reagent preparation. Including preparation of absolute ethanol and kit.

[0061] The prepared kit is Blood Genomic DNA Extraction Kit (Tiangen): before use, add absolute ethanol to the rinse solutions PW and GD, and add the volume according to the label on the bottle.

[0062] Step 2.2: Genomic DNA extraction from blood. Specifically, the following steps (1) to (11) may be included:

[0063] (1) Add 200 μL of blood to a 1.5 mL centrifuge tube.

[0064] (2) Add 20 μL Proteinase K solution and mix well.

[0065] (3) Add 200 μL buffer GB, mix thoroughly by inversion, bathe in 70°C water for 10 minutes (the solution should become clear), and centrifuge briefly.

[0066] (4) Add 200 μL of absolute ethanol, shake and mix well for 15 seconds (flocculent precipitation may appear at this time), and briefly centrifuge.

[0067] (5) Add the solution and flocculent precip...

Embodiment 3

[0082] The PCR product amplification method may comprise the following steps:

[0083] Step 3.1: Use the genomic DNA obtained in step 2.2 and 15 CAP samples from 2016-2018 as DNA templates, and use the primers synthesized in step 1.2 to configure a PCR reaction system. The specific composition of this reaction system is shown in Table 2 below.

[0084] Table 2

[0085] Reagent components volume 2×KOD FX Buffer 25 μL 2.0mM dNTPs 10μL HTT-F (10uM) 1.5μL HTT-R (10uM) 1.5μL KOD FX enzyme 1μL dna 1μL DI water 11μL

[0086] The CAP (COLLEGE OF AMERICAN PATHOLOGISTS, College of American Pathologists) laboratory was established in 1946 and is widely recognized as a leader in laboratory quality assurance. The implementation method of CAP external quality assessment is that the organizer provides a set of samples to be tested to the participating laboratories, and the participating laboratories complete the testing of th...

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Abstract

The invention provides a method and a primer pair for detecting a CAG trinucleotide repetition sequence of an HTT (huntingtin) gene. The method comprises the steps of synthesizing the primer pair, extracting genome DNA (deoxyribonucleic acid) from a sample to be detected as a DNA template preparing a PCR (polymerase chain reaction) amplification system comprising the primer pair and an amplification template, performing amplification reaction on the PCR amplification system to form a PCR product containing the CAG trinucleotide repetition sequence, detecting the PCR product through capillary electrophoresis, and calculating a CAG trinucleotide repetition number of the HTT gene of the sample to be detected according to an electrophoresis detection result. The trinucleotide repetition sequence can be detected without sequencing.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a method and primer pair for detecting the CAG trinucleotide repeat sequence of HTT gene. Background technique [0002] Huntington's disease (HD), also known as Huntington's disease, is an insidious onset hereditary neurodegenerative disease characterized by chorea-like involuntary movements, mental disorders, and dementia. It is inherited in an autosomal dominant manner. Its pathogenicity is caused by the abnormal amplification mutation of the CAG trinucleotide located on the Huntington gene IT15 (interesting transcript 15) on the short arm of chromosome 4. [0003] HD is an autosomal dominant genetic disease, and the expression product of IT15 gene (also known as HD gene or HTT gene) is the polypeptide Huntingtin (HTT) protein with a size of about 3144 amino acids. Mutant huntingtin contains an expanded chain of glutamine residues, and the pathological changes are...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156
Inventor 冯薇翟瑞雪智慧芳倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD