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Kit and method for detecting whole exomes of ADAMTS13 gene

An all-exome and kit technology, applied in the fields of life sciences and biology, can solve the problems of large ADAMTS13 capacity, multiple mutation types, and prevent in-depth research on ADAMTS13 gene mutations, and achieve low cost, simple operation, and high sensitivity. Effect

Inactive Publication Date: 2017-06-20
杭州艾迪康医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the large capacity of ADAMTS13, many mutation types, and scattered distribution of mutation sites, in-depth research on ADAMTS13 gene mutations is prevented, and fluorescent quantitative PCR method is not applicable

Method used

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  • Kit and method for detecting whole exomes of ADAMTS13 gene
  • Kit and method for detecting whole exomes of ADAMTS13 gene
  • Kit and method for detecting whole exomes of ADAMTS13 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The primers for detecting the full exons of the ADAMTS13 gene include: 26 pairs of forward and reverse primers that amplify and cover the entire exons of the ADAMTS13 gene; the base sequence of the extended primers is:

[0032]

[0033]

[0034]

[0035]

[0036] The primers also include a pair of sequencing primers, the base sequence of which is

[0037] M13-F: TGTAAAACGACGGCCAGT

[0038] M13-R: AACAGCTATGACCATG

[0039] In the detection, first use the above 26 pairs of forward and reverse amplification primers to amplify the DNA fragments covering the entire exon of the ADAMTS13 gene to obtain the amplified product, and then use the above 1 pair of sequencing primers to sequence the amplified product , to obtain the gene sequence of the amplified product.

[0040] In terms of primer design, each pair of primers designed is located on both sides of the exon sequence to be amplified, that is, the amplified region includes the entire sequence of the exon. ...

Embodiment 2

[0048] (1) Extract the genomic DNA in the blood (operate according to the instructions of the blood DNA extraction kit (Tiangen Biology)):

[0049] 1) Take 500ul of blood and add 1000ul of red blood cell lysate, mix by inversion, and let stand at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 3000rpm for 5 minutes, suck off the supernatant, leave the white blood cell pellet, add 200ul buffer GA, shake until thoroughly mixed.

[0050] 2) Add 20 μl proteinase K solution and mix well.

[0051] 3) Add 200 μl buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0052] 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent sediment may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0053] 5) Add the solution and fl...

Embodiment 3

[0083] Eight clinical patient samples were taken, all of which were confirmed to be suffering from TTP, and whether the ADAMTS13 mutation existed in the eight samples was detected. The genome was extracted, reagents were prepared and tested according to the method described in Example 2. Add 2 μl of each sample into the detection system PCR reaction solution. At the same time, make a positive control, a negative control, and a blank control. The test results are as follows:

[0084] The sequencing results of the positive control were compared with the wild-type ADAMTS13 sequence (GeneBank No.: NG_011934.2), and it was found that the two were completely consistent, which proved that the primers used could accurately amplify all the exons of the ADAMTS13 gene. The method of amplification and sequencing is accurate and reliable, and meets the requirements of gene amplification and sequencing.

[0085] The negative control substance and the blank control substance did not produ...

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Abstract

The invention discloses a kit and a method for detecting whole exomes of the ADAMTS13 gene. The kit comprises 26 pairs of forward primers and reverse primers for amplifying and covering whole exome sequences of the ADAMTS13 gene, wherein a segment of M13-F primer sequence with the length being 18bp is added at the 5' terminal of an upstream primer of PCR amplification, and a segment of M13-R primer sequence with the length being 16bp is added at the 5' terminal of a lower primer of PCR amplification. Based on the Sanger sequencing method, 1 pair of universal primers M13 is adopted as the sequencing primers, then the mutation condition of the whole exome sequences of the ADAMTS13 gene is detected, and the kit is used for genetic diagnosis for patients suffering from thrombotic thrombocytopenic purpura.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and in particular relates to a kit and a method for detecting the mutation of the whole exon of ADAMTS13 gene. Background technique [0002] In 1924, Eli Moschcowitz initially detailed thrombotic thrombocytopenic purpura (thromboticthrombocytopenicpurpura, TTP), which is mainly manifested clinically as a typical triad: thrombocytopenia, microangiopathic hemolytic anemia, and nervous system damage; if at the same time Accompanied by kidney damage and fever, the classic "five syndromes" of TTP are formed. In 1997, the link between very large molecular weight vWF multimers in the remission plasma of patients with chronic relapsing TTP and the absence of vWF lyase was formally established. Afterwards, the researchers purified this proteolytic enzyme and performed genome-wide linkage analysis on congenital TTP family members. The ADAMTS13 gene is located at 9q34, with a length of 37kb a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2531/113C12Q2535/101
Inventor 李文静刘赵玲王淑一
Owner 杭州艾迪康医学检验中心有限公司
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