Primer and method for quickly detecting IT15 gene CAG repeat sequence dynamic mutation

A repeat sequence and gene technology, applied in the field of biomedicine, can solve the problems of high experimental conditions, difficulty in popularizing and popularizing gene mutation detection technology, and difficulty in clinical application in general hospitals.

Inactive Publication Date: 2008-01-23
SUZHOU UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the use of primers and conditions reported in foreign literature, it is difficult to amplify the target band, and the isotope operation requires high experimental conditions, and the isotope operation is risky and complicated. Therefore, the existing Huntington's disease IT15 gene mutation Detection technology is difficult to promote and popularize, which brings difficulties to ordinary hospitals for clinical application

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
  • Primer and method for quickly detecting IT15 gene CAG repeat sequence dynamic mutation
  • Primer and method for quickly detecting IT15 gene CAG repeat sequence dynamic mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: A primer for rapidly detecting the dynamic mutation of the CAG repeat sequence of the IT15 gene, consisting of a first pair of primers corresponding to the first step of PCR amplification and a second pair of primers corresponding to the second step of PCR amplification;

[0077] The first pair of primers adopts any one pair of primers in the following four pairs of primers:

[0078] (I), exogenous sense: 5'CTTGCTGTGTGAGGCAGAACCTG-3'

[0079] Exogenous antisense: 5'GGCTGAGGAAGCTGAGGAG-3'

[0080] (II), exogenous sense: 5'TTCACACACAGCTTCGC-3'

[0081] Exogenous antisense: 5'CCTCCTCTCGAAGTGTCCCG-3'

[0082] (III), exogenous sense: 5'GGCAGAGTCCGCAGGCTAG-3'

[0083] Exogenous antisense: 5'AAGTTCCATAGCGATGCCCAG-3'

[0084] (IV), exogenous sense: 5'CTAGGGCTGTCAATCATGCTGG-3'

[0085] Exogenous antisense: 5'AAGTTCCATAGCGATGCCCAG-3'

[0086] The second pair of primers adopts any pair of primers in the following two pairs of primers:

[0087] (1), endogenous sen...

Embodiment 2

[0092] Embodiment 2: A method for rapidly detecting the dynamic mutation of the CAG repeat sequence of the IT15 gene, which is characterized in that it comprises the following steps:

[0093] Step 1: Prepare DNA

[0094] (1) Take blood samples from the human body;

[0095] (2) Obtaining DNA from blood samples, i.e. blood sample genomic DNA sample preparation:

[0096] Reagent preparation:

[0097] Anticoagulant: 0.48g citric acid, 1.32g sodium citrate, 1.47g glucose per 100ml;

[0098] Red blood cell lysate: 10mmol / L Tris-HCl, pH7.6;

[0099] 5mmol / L MgCl 2 ;

[0100] 10mmol / L NaCl;

[0101] White blood cell lysate: 10mmol / L Tris-HCl, pH7.6;

[0102] 10mmol / LEDTAPH8.0

[0103] 50mmol / L NaCl

[0104] 10mg / ml proteinase K (Protease K): 10mg Protease K dissolved in 1ml ddH 2 O. Aliquot and store at -20°C. When in use, melt at 4°C.

[0105] Procedure for obtaining DNA from a blood sample:

[0106] ①. Take 3ml of fresh blood and add 0.5ml of anticoagulant, mix well, c...

Embodiment 3

[0150] Example 3: Using the method of the present invention to rapidly detect the dynamic mutation of the IT15 gene CAG repeat sequence in a Huntington's disease family in Anhui Province.

[0151] 1. Materials and methods

[0152] 1.1 Research objects: A HD family in Anhui Province includes 4 generations of members, 11 of which were sampled and analyzed. 3ml of each peripheral venous blood (anticoagulated with 3.8% sodium citrate). The red blood cells were lysed, and the white blood cells were obtained by centrifugation, and the genomic DNA in the white blood cells was extracted by conventional saturated sodium chloride method.

[0153] 1.2 Amplify part of IT15 gene containing CAG repeat sequence. Using the first-step PCR primer (I) and the second-step PCR primer (I) mentioned in this patent, the htt gene of a chorea family member in Anhui Province was amplified according to the method in Example 2.

[0154] Amplification was performed using NEST-PCR. 1st PCR: The amplific...

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 provides the primer and method for fast detection of chorea IT15 gene CAG repeat sequence expansion mutation, characterized in that a specially designed primer is employed, and PCR expansion is carried twice to expand DNA amount to gel electrophoresis observable degree, finally gel electrophoresis is utilized to directly observe the situation of IT15 gene CAG repeat sequence dynamic mutation.

Description

technical field [0001] The invention relates to clinical detection technology in the field of biomedicine, in particular to a primer and a method for rapidly detecting the dynamic mutation of the IT15 gene CAG repeat sequence, and the detection result can be used for clinical auxiliary diagnosis of Huntington's disease. Background technique [0002] Huntington's disease (HD) is a neurodegenerative disease mainly affecting motor function, which is an autosomal dominant genetic disease. Symptoms of HD usually appear after the age of 45, and the main symptoms are uncontrolled large body movements, accompanied by cognitive impairment and mental abnormalities. The mutated gene IT15 that causes the disease was discovered in 1993. The gene is located on human chromosome 4, and it encodes a protein with a molecular weight of 350 kD, which is named Huntingtin (Htt). The IT15 gene consists of 67 exons, and there is a trinucleotide CAG repeat sequence after the 17th codon downstream ...

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): C12Q1/68
Inventor 秦正红林芳王进
Owner SUZHOU UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap