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Method, kit and specific primer for detecting CLCN1 (chloride channel 1) gene mutation

A specific and kit-based technology used in the detection of coding region and intron splice site mutations of CLCN1 gene

Active Publication Date: 2011-08-17
BGI GENOMICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are no reports in the art about the simultaneous determination of multiple mutations in the CLCN1 gene using multiple specific primer pairs

Method used

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  • Method, kit and specific primer for detecting CLCN1 (chloride channel 1) gene mutation
  • Method, kit and specific primer for detecting CLCN1 (chloride channel 1) gene mutation
  • Method, kit and specific primer for detecting CLCN1 (chloride channel 1) gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Design of specific primers

[0033] Design amplification primers according to the characteristics of CLCN1 exon distribution:

[0034] 1) According to the reference sequence of CLCN1 normal gene in NCBI and other databases, design primers of 15-30 BP according to the principle of complementary base pairing at the upstream of the 5' end and downstream of the 3' end of the mutation site. The primers should be designed in the conserved region of the nucleic acid series and have specificity;

[0035] 2) The product cannot form a secondary structure, the bases should be randomly distributed, and the primer itself cannot have 4 consecutive bases complementary;

[0036] 3) Design primers for 40-50bp extensions at both ends of each exon, including intron splicing sites (including mutation sites, which can cause frame-shift mutations).

[0037] 4) The annealing temperature difference between forward and reverse primers of all PCR fragments shall not exceed 5°C, and ...

Embodiment 2

[0041] Embodiment 2: sample DNA extraction

[0042] The positive sample used in the present invention is from a subject from Shandong, who was diagnosed as congenital myotonia by a hospital, sporadic cases without family history, and obtained blood samples after informed consent.

[0043] The QIAamp DNA Blood MINI Kit kit (Cat. No. 51106) from QIAGEN was used to extract DNA from the whole blood sample of the subjects. Exemplary steps are as follows:

[0044] (1) Add 20 μl proteinase K to a 1.5ml centrifuge tube;

[0045] (2) Add 200 μl whole blood sample to the centrifuge tube;

[0046] (3) Add 200 μl buffer AL to the centrifuge tube, and vortex for 15 seconds;

[0047] (4) 56 ° water bath for 15 minutes;

[0048] (5) Centrifuge properly so that all the liquid falls to the bottom of the centrifuge tube;

[0049] (6) Add 200 μl of absolute ethanol to the centrifuge tube, shake for 15 seconds to mix, and centrifuge properly to make all the liquid drop to the bottom of the c...

Embodiment 3

[0057] Embodiment 3: PCR amplification

[0058] The DNA sample extracted in Example 2 was amplified using the PCR amplification primer pair designed in Example 1, and the reaction used a 25 μl system, wherein: enzymes and buffer solutions were purchased from KATARA Company.

[0059]

[0060] The PCR conditions are:

[0061] Pre-denaturation at 95°C for 5 minutes

[0062] 94℃30sec→specific annealing temperature 30sec→72℃30sec (35 cycles)

[0063] Then extend for another 10 minutes.

[0064] Wherein, the specific annealing temperature of each pair of primers is as follows:

[0065]

[0066]

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PUM

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Abstract

The invention relates to a method, a kit and a specific primer for detecting CLCN1 (chloride channel 1) gene mutation. Specifically, the invention relates to the method, the kit and the specific primer for detecting the coding region and the intron shearing locus mutation of the CLCN1 gene.

Description

technical field [0001] The invention relates to a method, a kit and specific primers for detecting CLCN1 gene mutation. Specifically, the present invention relates to a method, a kit and specific primers for detecting mutations in coding regions and intron splicing sites of CLCN1 gene. Background technique [0002] Eukaryotic genes are composed of protein-coding exons (exons) and intervening sequence introns (introns) that do not code proteins. Exons are parts of eukaryotic genes that are preserved after splicing and can be expressed as proteins during protein biosynthesis. The exon is the gene sequence that appears in the mature RNA at the end, also known as the expression sequence, it is a nucleotide sequence that exists in both the initial transcription product and the mature RNA molecule. [0003] Genomic DNA is amplified by PCR method, and the obtained target fragment is sequenced, and compared with the normal sequence, the information of gene mutation can be obtained...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 魏晓明王金明
Owner BGI GENOMICS CO LTD