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Detection primers, kits and methods for htt and jph3 genes

A kit and primer pair technology, applied in the field of biomedical detection and diagnosis, can solve the problems of CAG repeat sequence instability and incompletely clear mechanism

Active Publication Date: 2022-07-01
BEIJING GRANDOMICS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mechanism of this phenomenon is not yet fully understood, and it may be related to the instability of the CAG repeat sequence during spermatogenesis

Method used

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  • Detection primers, kits and methods for htt and jph3 genes
  • Detection primers, kits and methods for htt and jph3 genes
  • Detection primers, kits and methods for htt and jph3 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 DNA extraction and PCR amplification 1. DNA extraction experimental method

[0060] Use a kit to extract genomic DNA from blood, for example, use the TIANamp Blood DNA Kit Blood Genomic DNA Extraction Kit (DP348), and the operation is performed according to the kit instructions:

[0061] 1. Add Buffer CL to the blood sample, mix well and centrifuge;

[0062] 2. Discard the supernatant, add Buffer GS, mix well and centrifuge;

[0063] 3. Add Buffer GB and Proteinase K, mix well and incubate;

[0064] 4. Let stand at room temperature, add Buffer BD, and mix well;

[0065] 5. Pass through the adsorption column CG2, stand at room temperature, centrifuge, and discard the filtrate;

[0066] 6. Add Buffer BD to the adsorption column CG2, centrifuge, and discard the filtrate;

[0067] 7. Add Buffer PW to the adsorption column CG2, centrifuge, and discard the filtrate;

[0068] 8. Repeat step 7;

[0069] 9. Centrifuge and dry the adsorption column CG2;

[0070] ...

Embodiment 2

[0088] Example 2 Using PacBio sequal for third-generation sequencing

[0089] 1. Use the library building kit to build the library

[0090] 1 Repair the mixed library

[0091] Repair solution preparation:

[0092]

[0093] Mix well, centrifuge, put into PCR thermal cycler, and carry out repair reaction. The specific conditions are as follows:

[0094]

[0095]

[0096] 2 connector connection

[0097] The connection solution system is as follows:

[0098]

[0099] Mix well, centrifuge, and carry out the ligation reaction in a PCR thermal cycler. The specific conditions are as follows:

[0100]

[0101] 3 Purification

[0102] Use AMPure XP magnetic beads for purification, and finally use double-distilled water to elute the magnetic beads, and store at -20°C.

[0103] 2. Sequencing

[0104] 3. Bioinformatics analysis of sequencing data

[0105] 1.call ccs reads: apply the pacbio platform Smrtlink5-1 analysis process to call ccs reads.

[0106] 2. Barcode sp...

Embodiment 3

[0109] Example 3 Using the PromethION platform of Oxford Nanopore Technology for third-generation sequencing 1. Building a library using a library building kit

[0110] 1Library preparation

[0111] 1.1 End repair and A-tail ligation

[0112] Take the DNA, put it on ice, add NEB end repair and A-tail ligation reagents, and mix. Incubate for 40 minutes at 20°C and 20 minutes at 65°C.

[0113] 1.2 Magnetic bead purification

[0114] Add 1×AMPure beads to the DNA, incubate at room temperature for 15 minutes, adsorb on a magnetic stand at room temperature for 5 minutes, and discard the supernatant.

[0115] Add 80% ethanol, adsorb on a magnetic stand, discard the supernatant, and repeat once. Dry at room temperature.

[0116] Add Ultra Pure Water and elute by pipetting at 37°C.

[0117] Let stand on a magnetic stand for 5 min, and aspirate the supernatant, which is the purified DNA.

[0118] 1.3 Ligation of sequencing adapters

[0119] Add NEB T4DNA quick ligation buffer, ...

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Abstract

The invention relates to the field of biomedical detection and diagnosis, in particular to a detection primer, a kit and a method for HTT and JPH3 genes. The primers and kits provided by the present invention, combined with the third-generation sequencing method, can effectively detect the dynamic mutation and / or interrupted repetition of the repetitive sequence of Huntingtin and Junctophilin-3 genes.

Description

technical field [0001] The invention relates to the field of biomedical detection and diagnosis, in particular to a detection primer, kit and method for HTT and JPH3 genes. Background technique [0002] Huntington disease (HD) is a late-onset autosomal dominant neurodegenerative disorder. The main pathological changes were selective neuronal loss in the striatum and cerebral cortex. The disease usually occurs in middle age (30-40 years old), and the average survival time after the onset is about 15 years [1] . Clinically, it is often divided into two types: adult type (adultonset HD) and juvenile type (juvenile HD). The incidence of juvenile type is less than 20 years old, accounting for only 10%. The juvenile type has a rapid progression and a short life cycle, usually 8-10 years. [2] . Adult-onset HD is characterized by involuntary dance-like movements, personality changes, and dementia. In the juvenile form, bradykinesia, rigidity, dystonia and epileptiform seizures...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 贺希文高玉梅郎娜梁帆王洋汪德鹏
Owner BEIJING GRANDOMICS BIOTECH
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