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Primer set for detecting human SERPINF1 gene mutation and kit thereof

A kit and genome technology, applied in the field of primer sets and kits for detecting human SERPINF1 gene mutations, can solve problems such as missing test results, failure to detect large fragment deletions, copy number variations, and no clear candidate genes, etc., to achieve Avoid omissions, save costs and manpower, and ensure accuracy

Inactive Publication Date: 2020-09-04
SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has no advantages for the detection of no clear candidate genes, a large number of candidate genes, and a large number of samples
At the same time, for Sanger sequencing, the sequencing fragments are short, and mutation types such as large fragment deletions and copy number variations cannot be detected, resulting in omission of detection results

Method used

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  • Primer set for detecting human SERPINF1 gene mutation and kit thereof
  • Primer set for detecting human SERPINF1 gene mutation and kit thereof
  • Primer set for detecting human SERPINF1 gene mutation and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] The DNA sequence of the SERPINF1 gene (NG_028180.1) was analyzed according to the NCBI GeneBank database, and the amplification primers for exons 1 to 8 were designed. The amplified region includes all exons of SERPINF1 and the intron sequence at the junction of exons and introns not less than 50 bp. The nucleotide sequences of primers used in PCR amplification are shown in Table 1.

[0064] Table 1 SERPINF1 gene exon primer amplification and sequencing sequence

[0065]

[0066]

Embodiment 2

[0068] A kit for detecting SERPINF1 gene mutation, comprising:

[0069] The primer set for PCR amplification from the 1st to the 8th exon designed in Example 1 for detecting the mutation of the SERPINF1 gene;

[0070] PCR amplification reagents include: 10mM dNTP Mix, containing Mg 2+ 2×Phanta Max Buffer, Phanta MaxSuper-Fidelity DNA Polymerase and de-RNase and DNase enzyme water (DEPC water); the working concentration of each component in the PCR amplification system is: 10000μM dNTP Mix / 2mM 2×Phanta Max Buffer / 1U / μl Phanta MaxSuper-Fidelity DNA Polymerase, the upstream and downstream primers are 5μM and the template concentration is 50-400ng / μl.

[0071] PCR product purification reagents include: 1U / μl SAP enzyme, 10U / μl ExoI enzyme, RNase and DNase-free water; the working concentration of each component is: 0.05U / μl SAP enzyme, 0.5U / μl ExoI enzyme.

[0072] DNA sequencing reagents include: the above sequencing primer set for detecting SERPINF1 gene mutation, BigDye 3.1 m...

Embodiment 3

[0076] Utilize the kit for detecting the mutation of the SERPINF1 gene in the above-mentioned embodiment 2 to carry out the detection method of the mutation of the human SERPINF1 gene, the steps are as follows:

[0077] (1) Extraction of genomic DNA from peripheral blood

[0078]Collect 5ml of peripheral blood from the patient, use Omega Company DNA Extraction Kit (D3392-01) to extract whole blood genomic DNA, and use NanoDrop 2000 / 2000c spectrophotometer to measure DNA concentration and purity; see the product manual for specific operating steps and conditions;

[0079] (2) PCR amplification

[0080] The PCR reaction system is 40 μl, including:

[0081] 10mM dNTP Mix 1μl, 2×Phanta Max Buffer 20μl, Phanta Max Super-Fidelity DNA Polymerase 1μl, upstream primer 4μl, downstream primer 4μl, DNA template 50ng~400ng, make up to 40μl with DEPC water.

[0082] Use the PIC-200 PCR instrument of BIO-RAD Company to perform touchdown PCR. The PCR reaction conditions are as follows: 95°C...

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Abstract

The invention provides a primer set for detecting a human SERPINF1 gene mutation and a kit thereof. The invention belongs to the technical field of gene detection. The kit for detecting the human SERPINF1 gene mutation comprises a PCR amplification primer set which is used for detecting the SERPINF1 gene mutation and consists of first to eighth exons, a PCR amplification reagent, a PCR product purification reagent and a sequencing reagent containing the sequencing primer set for detecting the SERPINF1 gene mutation. The kit provided by the invention carries out mutation detection for the firstto eighth exons of an SERPINF1 gene and ensures accuracy and specificity of a measurement result. The primer set can be used for detecting the SERPINF1 gene mutation of a VI type osteogenesis imperfect (OI) patient, and the SERPINF1 gene mutation comprises a homozygous mutation, a compound heterozygous mutation and the like. Meanwhile, the primer set can also be used for screening VI type OI carriers and detecting complex disease lesions possibly caused by the gene mutation.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a primer set and a kit for detecting human SERPINF1 gene mutation. Background technique [0002] Osteogenesis imperfecta (OI) is a rare hereditary connective tissue disease caused by collagen synthesis disorder. The main clinical phenotypes include osteoporosis and increased bone fragility, blue sclera, dentin insufficiency, and premature otosclerosis. Osteogenesis imperfecta has genetic heterogeneity, including autosomal dominant inheritance, autosomal recessive inheritance, and X-linked inheritance. More than 80% of patients have mutations in the type I collagen structural gene COLIAl or COLIA2. Caused by autosomal dominant inheritance. Osteogenesis imperfecta type VI is an autosomal recessive disorder caused by mutations in the SERPINF1 gene. The protein encoded by the SERPINF1 gene is pigment epithelium-derived factor (PEDF), which is a secreted glycopro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883
CPCC12Q1/6883C12Q2600/156
Inventor 鲁艳芹扈瑞平魏玲张磊亮韩金祥任秀智王延宙张更林彭传明
Owner SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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