Method and primer for detecting hereditary blood coagulation factor XI(F11) genes

A technology of coagulation factor, F11E1R, applied in the field of medical detection, can solve problems such as easy bleeding

Inactive Publication Date: 2017-03-22
FUZHOU ADICON CLINICAL LAB INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, patients with lower FXI levels may be more prone to bleeding, especially after surgic

Method used

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  • Method and primer for detecting hereditary blood coagulation factor XI(F11) genes
  • Method and primer for detecting hereditary blood coagulation factor XI(F11) genes
  • Method and primer for detecting hereditary blood coagulation factor XI(F11) genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] A primer for detecting the polymorphic mutation site of coagulation factor XI (F11) gene. The design of the primer is an amplification primer designed for the whole exon of coagulation factor XI (F11), including:

[0128] The primers for amplifying the whole exon sequence of coagulation factor XI (F11) gene have the base sequence:

[0129] F11 E1 F: TGTAAAACGACGGCCAGTAGGCACACAGGCAAAATC

[0130] F11 E1 R: AACAGCTTGACCATGGATATTAGCGGAACATCTCTAC

[0131] F11 E2 F: TGTAAAACGACGGCCAGTACACAGTCACACTAAGGAAT

[0132] F11 E2 R: AACAGCTATAGACCATGCTCTCCCAGCCCATAGTT

[0133] F11 E3 F: TGTAAAACGACGGCCAGTGCTACTTGCCTTGCCTT

[0134] F11 E3 R: AACAGCTATAGACCATGGTTAAGAATAGCAATCTCCC

[0135] F11 E4 F: TGTAAAACGACGGCCAGTGCTTTCTGTGTGCTGACT

[0136] F11 E4 R: AACAGCTTGACCATGCGATGTGGCGATATGTGTA

[0137] F11 E5 F: TGTAAAACGACGGCCAGTCTAGAATCTGGAAGGTACTCAT

[0138] F11 E5 R: AACAGCTATAGACCATGGGCATAAAGTTGATGGCAAA

[0139] F11 E6 F: TGTAAAACGACGGCCAGTCAGGTCCTCTCTCCAAAG

[0140] F11 E6 R: AACAGCTTGACCATGGCTGGTATCCTGAG...

Embodiment 2

[0168] Operation process of blood / cell / tissue genomic DNA extraction kit (Tiangen Bio):

[0169] (1) Extract tissue DNA from blood: 1) Draw 300 μl of blood and add 900 μl of red blood cell lysate, mix upside down, place it at room temperature for 5 minutes, and mix upside down several times during the process. Centrifuge at 12,000 rpm for 1 min, aspirate the supernatant, leave the white blood cell pellet, add 200 μl of buffer GA, and shake until thoroughly mixed. 2) Add 20μl proteinase K solution and mix well. 3) Add 200μl of buffer GB, fully invert and mix well, place at 70°C for 10 minutes, the solution should be clear, and centrifuge briefly to remove the water droplets on the inner wall of the cap. 4) Add 200μl of absolute ethanol, shake and mix well for 15 seconds. At this time, flocculent precipitation may appear. Centrifuge briefly to remove water droplets on the inner wall of the cap. 5) Add the solution and flocculent precipitate obtained in the previous step to an ads...

Embodiment 3

[0224] In order to verify the feasibility of the primers and the entire detection system, 15 clinical blood samples (numbered 1-15) were taken. Follow the reagents and methods of Examples 1 and 2 to extract whole genome DNA, prepare reagents, amplify and sequence. Add 1μl of the sample to the PCR reaction solution of the detection system. The results of electrophoresis are as figure 2 It is shown that the primers of the present invention can effectively amplify blood samples and have a single band.

[0225] Sample Exon (1-15) amplification product Sequencing results Point mutation Extra gene insertion Ploidy 1 Clear and single band Single peak without clutter no no no 2 Clear and single band Single peak without clutter no no no 3 Clear and single band Single peak without clutter no no no 4 Clear and single band Single peak without clutter no no no 5 Clear and single band Single peak without clutter no no no 6 Clear and single band Single peak without clutter ...

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Abstract

The invention discloses a method and primer for detecting gene mutation relevant to the hereditary blood coagulation factor XI(F11). The primer comprises 15 pairs of primers with amplification detecting all-exon sequences. The Sanger sequencing technology and the sequencing primers are adopted. Hereditary blood coagulation factor XI(F11) deficiency disease all-exon and relevant mutation can be fast detected. The detecting result completed through the method and primer is accurate, diagnosis of the hereditary blood coagulation factor XI(F11) deficiency disease can be assisted, and the method and primer have important reference significance on early intervention, early treatment and antenatal diagnosis.

Description

Technical field [0001] The invention belongs to the field of medical detection, and particularly relates to primers and methods for detecting genetic mutations related to hereditary coagulation factor XI (F11). Background technique [0002] The coagulation factor XI (F11) gene is located on the long arm of chromosome 4 (4q35), with a total length of 23kb, containing 15 exons and 14 introns (A~N). Exon 1 is encoded as 5′ non- In the translation region, exon 2 encodes a signal peptide of 18 amino acids, exon 3 to exon 10 encode mature protein, and a spherical domain formed by four tandem repeats of 90 or 91 amino acids at the amino terminus, in which Every two exons encode a tandem repeat, and each tandem repeat encodes 6 conserved cysteines (Cys), and the positions of the introns between them in these 4 tandem repeats are basically the same. Exons 11-15 are encoded as the carboxyl end of the polypeptide chain, separated by 5 introns, of which the last 4 introns are located in the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 黄开新孙翠莲王淑一
Owner FUZHOU ADICON CLINICAL LAB INC
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