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Method for detecting mutation of F13A1 gene, kit, oligonucleotide and application of oligonucleotide

An oligonucleotide and kit technology, used in biological science and biology, can solve problems such as instability of blood clots, and achieve the effects of reducing cumbersomeness, reducing detection costs, and simplifying sequencing steps

Inactive Publication Date: 2019-04-09
南京艾迪康医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Deficiency of clotting factor 13 leads to unstable clots

Method used

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  • Method for detecting mutation of F13A1 gene, kit, oligonucleotide and application of oligonucleotide
  • Method for detecting mutation of F13A1 gene, kit, oligonucleotide and application of oligonucleotide
  • Method for detecting mutation of F13A1 gene, kit, oligonucleotide and application of oligonucleotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] An oligonucleotide for detecting the polymorphic mutation site of the F13A1 gene, its base sequence is:

[0062] F13A1-1F: TGTAAAACGACGGCCAGTCCAGCAACTGGTTGCGGT;

[0063] F13A1-1R:AACAGCTATGACCATGCCCCATTTGGTGGCATTCTA

[0064] F13A1-2F: TGTAAAACGACGGCCAGTTGCTGTAGGATTATGGAGGGTG;

[0065] F13A1-2R:AACAGCTATGACCATGGGCAGAACTGGGACAAGGCT;

[0066] F13A1-3F: TGTAAAACGACGGCCAGTAGTGCCTTGTGGTCTGAAACG;

[0067] F13A1-3R: AACAGCTATGACCATGACTGTGCCTGTACCCACCCTCT;

[0068] F13A1-4F: TGTAAAACGACGGCCAGTTGCAGACTTGCCTGATTTGTAT;

[0069] F13A1-4R: AACAGCTATGACCATGACCTCCAACTCCCGAACTCA;

[0070] F13A1-5F: TGTAAAACGACGGCCAGTAAAGAGCCAGAGTTCGTCAGC;

[0071] F13A1-5R: AACAGCTATGACCATGCGCAGTTGTCTTTTATGAGTCCC;

[0072] F13A1-6F: TGTAAAACGACGGCCAGTGGCCTGACTTTGGTAAATGATT;

[0073] F13A1-6R: AACAGCTATGACCATGACTGGCATATATACTGAGGCAAA;

[0074] F13A1-7F: TGTAAAACGACGGCCAGTTGGTAAACCGAACAGAAGTGG;

[0075] F13A1-7R: AACAGCTATGACCATGAATTCTATAGTGTCAACAGGGGC;

[0076] F13A1-8F: TGTAAAACGACGGCCAGTAACCC...

Embodiment 2

[0102] Example 2 Blood Sample DNA Extraction

[0103] Blood sample DNA extraction (according to the instructions of Tiangen Bio-Blood / Cell / Tissue Gene DNA Extraction Kit): extract human blood sample DNA, the specific extraction method is as follows:

[0104] (1) Take 300 μl of blood and add 900 μl 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 12000rpm for 1min, suck off the supernatant, leave the white blood cell pellet, add 200μl buffer GA, shake until thoroughly mixed;

[0105] (2) Add 20 μl proteinase K solution and mix well;

[0106] (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;

[0107] (4) Add 200 μl of absolute ethanol, shake and mix well for 15 seconds. At this time, flocculent sediment may appear, and bri...

Embodiment 3

[0114] Example 3 sample DNA amplification

[0115] The blood sample DNA extracted according to Example 2 was then amplified with the amplification primers in Example 1 to amplify the exon of the human F13A 1 gene to obtain the amplified product. Amplification was carried out on a conventional PCR instrument, available instruments include ABI veriti (Applied Biosystems, USA) and the like. The details are as follows:

[0116] (i) According to the number of samples n (number of samples = number of samples to be tested + 1 negative control + 1 positive control + 1 blank control), the PCR amplification reaction solution of the detection system is taken, and 19 μl of each tube is divided into reaction tubes;

[0117] (ii) Add 1 μl of the above-mentioned processed sample to be tested, the negative control substance, and the positive control substance into the reaction tube respectively, mix well, centrifuge at low speed for several seconds, perform PCR amplification, and obtain the ...

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PUM

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Abstract

The invention discloses a primer and a method for detecting the mutation of an F13A1 gene of a patient with blood coagulation factor deficiency. The primer comprises a primer amplifying the sequence of a whole exon of the F13A1 gene; and a Sanger sequencing technique and a sequencing primer are adopted. The mutation of the whole exon of the F13A1 gene in the body of the patient with blood coagulation factor deficiency is detected rapidly. The detection result obtained by using the method is accurate, the blood coagulation factor deficiency is assisted to be diagnosed, and the method is of great reference significance for predicating the risk of the disease and customizing a therapeutic schedule in a targeting way.

Description

technical field [0001] The invention belongs to the field of biological science and biotechnology, and in particular relates to a F13A1 gene mutation method, kit, oligonucleotide and application thereof. Background technique [0002] Coagulation factors are a class of protein components involved in the blood coagulation process. Their physiological role is to be activated when blood vessels are bleeding, and stick together with platelets, thereby blocking the leaks on blood vessels. According to the order in which coagulation factors were discovered, coagulation factors are named as coagulation factors 1-13, among which coagulation factor 13 is the last factor involved in the coagulation process, has the activity of transglutaminase, and plays a role in stabilizing fibrin clots effect. A deficiency of clotting factor 13 will cause the clot to become unstable. [0003] Coagulation factor 13 is composed of two different pairs of peptide chains non-covalently linked, and the...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6869C12N15/11
CPCC12Q1/6869C12Q1/6883C12Q2600/156C12Q2531/113C12Q2525/191
Inventor 林筱剑吴鹏飞王淑一
Owner 南京艾迪康医学检验所有限公司