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Method and primer for detecting whole exons of FIX gene

An all-exon, FIX-2 technology, applied in the fields of life science and biology, can solve the problems of large FIX capacity, high mutation rate, and many mutation types, and achieve the effect of simplifying the operation steps.

Inactive Publication Date: 2016-04-20
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 FIX, many types of mutations, high mutation rate, and scattered distribution of mutation sites, in-depth research on FIX gene mutations is prevented, and fluorescent quantitative PCR method is not applicable.

Method used

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  • Method and primer for detecting whole exons of FIX gene
  • Method and primer for detecting whole exons of FIX gene
  • Method and primer for detecting whole exons of FIX gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Detect primers for detecting the entire exon of the FIX gene, including: 7 pairs of forward and reverse primers that amplify and cover the entire exon of the FIX gene; the base sequence of the extended primer is:

[0076] FIX-1-F: TGTAAAACGACGGCCAGTGTACAACTAATCGACCTTACCACTT

[0077] FIX-1-R: AACAGCTATGACCATGCGTGCTGGCTGTTAGACTCT

[0078] FIX-2+3-F: TGTAAAACGACGGCCAGTCCCTAAAGAGAAATTGGCTTTCAG

[0079] FIX-2+3-R: AACAGCTATGACCATGGAATTGCTTACCAACATACTGCTTC

[0080] FIX-4F: TGTAAAACGACGGCCAGTTGGCTTCCAGGTCAGTAGTT

[0081] FIX-4R: AACAGCTATGACCATGAGGTGAGTCGGAACATCATTATTAC

[0082] FIX-5F: TGTAAAACGACGGCCAGTCACTCTTTATTTCAAGGCTCCAA

[0083] FIX-5R: AACAGCTATGACCATGAAGGAAGCAGATTCAAGTAGGA

[0084] FIX-6F: TGTAAAACGACGGCCAGTGCTTGAGACTCTATTCACTGATTAG

[0085] FIX-6R: AACAGCTATGACCATGCATCCCAATAGGTCTGTCTAGTA

[0086] FIX-7F: TGTAAAACGACGGCCAGTGCCAGCACCTAGAAGCCAATA

[0087] FIX-7R: AACAGCTATGACCATGCCCTTCTGCCTTTAGCCCAAT

[0088] FIX-8F: TGTAAAACGACGGCCAGTGGTGAACATAATATTGAGGAGACAG...

Embodiment 2

[0119] The operation process of the blood DNA extraction kit (Tiangen Biology):

[0120] (1) Genomic DNA extracted from blood:

[0121] 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 5min, suck off the supernatant, leave the white blood cell pellet, add 200uL buffer GA, shake until thoroughly mixed.

[0122] 2) Add 20 μl proteinase K solution and mix well.

[0123] 3) Add 200 μl of 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.

[0124] 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0125] 5) Put the solution and flocculent prec...

Embodiment 3

[0152] Take 7 samples of clinical patients, all of which are not sure whether they have HB, and detect whether there is a FIX mutation in the 7 samples. The genome was extracted, reagents were prepared and tested according to the method described in Example 2. Add 2 μl of each sample to the detection system PCR reaction solution. At the same time, make positive, negative, and blank controls. Detect with common PCR instrument, the time is 160 minutes.

[0153] Partial forward sequencing results of No. 1 whole exon sequence of sample 1 are as follows: figure 1 As shown, it is wild type, and no FIX mutation was detected.

[0154] Part of the forward sequencing results of the No. 2 and No. 3 whole exon sequences of sample 2 are as follows: figure 1 As shown, it is wild type, and no FIX mutation was detected.

[0155] Partial forward sequencing results of No. 4 full exon sequence of sample 3 are as follows:figure 1 As shown, it is wild type, and no FIX mutation was detected. ...

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Abstract

The invention discloses a method, primer and a kit for detecting whole exons of an FIX gene. Each of the primer and the kit includes seven pairs of forward and reverse primers which are expanded to cover the whole exon sequence of the FIX gene; a pair of universal primers is utilized as sequencing primers for detecting mutation conditions of the whole exon sequence of the FIX gene by virtue of a Sanger sequencing method. The primer and the method can be applied to the aspects of gene diagnosis, family female carrier detection, prenatal gene diagnosis and the like of patients with hemophilia B.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, in particular to primers and methods for detecting FIX gene whole exon mutation Background technique [0002] Hemophilia B, also known as coagulation factor IX (FIX) deficiency, is an X-chromosome-linked recessive genetic disease. The pathogenesis is that mutations in the FIX (F9) gene lead to decreased plasma FIX content or functional defects. Males The incidence rate is 1 / 30000, and it is rare in women. The symptoms of hemophilia B are mainly manifested as excessive bleeding in various parts of the patient's body spontaneously or after injury. At present, there is no effective cure, and the symptoms can only be alleviated or delayed by blood transfusion or FIX concentrated preparations, which brings great harm to the patient's family and society. heavy burden. [0003] The FIX gene is located at Xq26.3-27.2, with a total length of 34kb, consisting of 8 exons, 7 introns and regula...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2535/101C12Q2527/143C12Q2531/113
Inventor 李文静林有升王淑一
Owner 杭州艾迪康医学检验中心有限公司
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