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Nucleic acid encoding F5 gene mutant and application thereof

A coding and nucleic acid technology, applied in the field of genetic engineering, can solve the problem that the detection method needs to be further studied, and achieve the effect of rapid detection method

Active Publication Date: 2019-03-22
福州福瑞医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the determination of the causative gene of hereditary factor V deficiency and the detection method of hereditary factor V deficiency still need further research

Method used

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  • Nucleic acid encoding F5 gene mutant and application thereof
  • Nucleic acid encoding F5 gene mutant and application thereof
  • Nucleic acid encoding F5 gene mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Determining the Causative Mutation of Inherited Factor V Deficiency

[0058] 1. Sample source

[0059] A 12-year-old female patient (proband) with gastrointestinal bleeding from Fujian Province, China, presented with black tarry stools for 1 day and was admitted to hospital. The proband usually stops bleeding after the skin is damaged, and easily leaves petechiae or ecchymosis after collision. The parents were married to non-consanguineous relatives, and neither of them had a history of bleeding. Laboratory tests revealed that their plasma prothrombin time (PT) was 54.3S (9.8-12.1S for the control), and activated partial thromboplastin time (APTT) was 168.7S (22.7S for the control). -31.8S), thrombin time (TT) 15.2S (control: 14.0-21.0S), coagulation factor V activity assay (FV:C) 1.2% (control: 60-150%), preliminarily diagnosed as coagulation factor V deficiency disease. The hemorrhagic pedigree as figure 1 As shown, the arrow points to the proband, the s...

Embodiment 2

[0073] Example 2 Sanger method sequencing verification

[0074] Detect the F5 gene of the family members (including 1 patient and 2 normal family members) in the family members of patients with hereditary factor V deficiency described in Example 1: c.587G>T and c for the F5 gene .Design primers for 5195C>T mutation, then obtain the relevant sequence of the mutation site by PCR amplification, product purification and sequencing, and verify whether the c.587G>T and c .5195C>T mutation and the correlation between inherited factor V deficiency.

[0075] The specific method steps are as follows:

[0076] 1. DNA extraction

[0077] Refer to the method for extracting DNA described in Example 1.

[0078] 2. Primer design and PCR reaction

[0079] First, refer to the human genome sequence database hg19 / build36.3, and design specific primers for the 5th and 15th exons where the c.587G>T and c.5195C>T mutations of the F5 gene are located, respectively. The specific sequences are as f...

Embodiment 3F5

[0089] Embodiment 3 F5 gene mutant detection kit

[0090] The detection kit for F5 gene mutants includes primers capable of detecting the c.587G>T mutation and c.5195C>T mutation of the F5 gene, and is used to screen susceptible biological samples. These primers are specific primers for the F5 gene, and their sequences are shown in SEQ ID NO: 3-6 in Example 2.

[0091] The specific steps for using the above kit to screen biological samples susceptible to hereditary factor V deficiency are: extract the DNA of the test subject according to the method described in step 2 of Example 1, and use the extracted DNA as a template to combine with the above-mentioned F5 gene Perform PCR reaction with specific primers, purify the PCR product according to conventional methods in the art, sequence the purified product, and then observe whether the sequence obtained by sequencing has c.587G>T and c.5195C>T mutations at the same time, can Effectively detect whether the F5 gene mutant exists ...

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Abstract

The invention discloses a nucleic acid encoding a F5 gene mutant and an application thereof, and belongs to the technical field of genetic engineering. This mutant has c. 587G)T mutation or c. 5195C)T mutation or c. 587G) T and c. 5195C) T complex heterozygous mutation compared with the wild type, the amino acid sequence of the encoded polypeptide has p. Gly196Val mutation or p. Ser1732Leu mutation or p. Gly196Val and p. Ser1732Leu complex mutation. By detecting the presence or absence of the new mutant, a biological sample susceptible to hereditary factor V deficiency can be effectively screened, and the detection method in the invention is fast, accurate, and efficient.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an F5 gene mutant and its application. In particular, the present invention relates to isolated nucleic acids encoding F5 mutants, isolated F5 mutant polypeptides, recombinant cells containing F5 mutant nucleic acids, and methods of screening biological samples for genetic factor V deficiency. Background technique [0002] Inherited factor V deficiency is a rare bleeding disorder with a prevalence of 1 in 1 million. Symptoms of the disease can begin at any age, but severe disease usually presents in childhood. Inherited factor V deficiency often causes epistaxis, bruising, bleeding under the skin, bleeding gums, prolonged or persistent bleeding after surgery, trauma, or excessive bleeding during childbirth. Women who lack coagulation factor V may have menorrhagia. In severe patients, the risks of hemarthrosis, intracranial hemorrhage, intrapulmonary hem...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N15/11C12N9/64C12N5/10C12Q1/6883C12Q1/02
CPCG01N33/5008C12N9/6424C12Q1/6883C12Q2600/156
Inventor 王开宇宋光运朱颜婷刘小霞
Owner 福州福瑞医学检验实验室有限公司
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