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Primers and kit for amplifying and detecting mutation of human COL1A1 and/or COL1A2 genes

A technology for sequencing primers and genes, which is used in DNA/RNA fragmentation, microbial determination/inspection, recombinant DNA technology, etc. It can solve the problems of low accuracy and specificity of determination results, incomplete detection results, and lack of amplification. , to save cost and manpower, reduce the number of PCR reactions, and simplify the amplification operation.

Pending Publication Date: 2020-08-18
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

However, because the full sequence of the COL1A1 or COL1A2 gene is very long, there is a lack of effective primers for amplifying the long fragments of the COL1A1 or COL1A2 gene. At the same time, the detection of the mutation site of the COL1A1 or COL1A2 gene in the prior art is usually the detection of a single mutation site, resulting in The test results are incomplete and have omissions, and the accuracy and specificity of the test results are not high.

Method used

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  • Primers and kit for amplifying and detecting mutation of human COL1A1 and/or COL1A2 genes
  • Primers and kit for amplifying and detecting mutation of human COL1A1 and/or COL1A2 genes
  • Primers and kit for amplifying and detecting mutation of human COL1A1 and/or COL1A2 genes

Examples

Experimental program
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Effect test

Embodiment 1

[0058] According to the NCBI GeneBank database COL1A1 (NG_007400.1) gene DNA reference sequence, design 2 pairs of long-segment PCR amplification primer pairs FP1 / RP1 and FP2 / RP2 to amplify the gene DNA sequence including all introns and exons of the gene . The exons covered by the two PCR products were 1-25 and 26-52, respectively, and the PCR amplification lengths were 7903bp and 7985bp, respectively. The PCR amplified fragments of each gene overlap each other by at least 150 bp to ensure that the subsequent upstream and downstream sequencing primers can detect each exon of the gene and its junction with the intron. For the exons included in the two long-fragment PCR amplifications, the upstream and downstream sequencing primers of the corresponding exons were designed respectively, and the sequencing primers were designed at the 60 bp upstream region at the junction of the exons and introns. Among them, there are 15 pairs of upstream and downstream sequencing sequences cor...

Embodiment 2

[0064] According to the DNA reference sequence of the COL1A2 (NG 007405.1) gene in the NCBI GeneBank database, three pairs of long-fragment PCR amplification primer pairs FP18 / RP18, FP19 / RP19, and FP20 / RP20 were designed to amplify all introns and exons of the gene. genetic DNA sequence. The exons covered by the three PCR products were 1-12, 13-34 and 35-52, respectively, and the PCR amplification lengths were 11549bp, 12218bp and 10502bp, respectively. The PCR amplified fragments of each gene overlap each other by at least 150 bp to ensure that the subsequent upstream and downstream sequencing primers can detect each exon of the gene and its junction with the intron. For the exons included in the three long-fragment PCR amplifications, the upstream and downstream sequencing primers of the corresponding exons were designed respectively, and the sequencing primers were designed at the 60 bp upstream region at the junction of the exons and introns. Among them, there are 31 pair...

Embodiment 3

[0071] A long fragment PCR amplification of human COL1A1 / COL1A2 gene DNA and a corresponding kit thereof, comprising:

[0072] 1) 2 pairs of primers FP1 / RP1 and FP2 / RP2 used for long fragment PCR amplification of COL1A1 gene in Example 1;

[0073] 2) 3 pairs of primers FP18 / RP18, FP19 / RP19 and FP20 / RP20 used in the long-fragment PCR amplification of the COL1A2 gene in Example 2;

[0074] 3) The PCR long fragment amplification reagent, including 10mM dNTP Mix, containing Mg 2+ 2×Phanta MaxBuffer, Phanta Max Super-Fidelity DNA Polymerase, RNase and DNase-free water; the working concentration of each component in the PCR amplification system is: 10000μM dNTP Mix, 2mM 2×Phanta Max Buffer, 1U / μlPhanta Max For Super-Fidelity DNA Polymerase, the upstream and downstream primers are both 5 μM, and the template concentration is 50-400 ng / μl.

[0075] A kit for detecting human COL1A1 / COL1A2 gene DNA mutation, comprising:

[0076] 1) The reagents for purifying long-segment PCR products...

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Abstract

The invention provides primers and a kit for amplification and mutation site detection of human COL1A1 and / or COL1A2 genes, and belongs to the technical field of gene detection. The kit for detectingthe DNA mutation of the human COL1A1 and / or COL1A2 genes comprises a sequencing primer group for detecting the mutation of the 1st to 52nd exons of the COL1A1 and COL1A2 genes. By adopting the specific primer group for mutation detection of the first exon to the 52nd exon of the COL1A1 gene and the COL1A2 gene, the accuracy and specificity of a measurement result are ensured; gene detection is carried out on hereditary diseases such as osteogenesis imperfectaI-IV type, Ehlers-Danlos syndrome, Caffey's disease, osteogenesis imperfectaand Ehlers-Danlos syndrome complications and the like causedby COL1A1 and COL1A2 gene mutation. The kit can be used for gene detection of complex diseases associated with COL1A1 and COL1A2 gene mutation.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to primers and kits for amplification and mutation detection of human COL1A1 and / or COL1A2 genes. Background technique [0002] Collagen is an important class of protein. With the development of biochemistry, molecular biology, and cell biology techniques, 27 different types of collagen have been discovered, which are called type I collagen, type II collagen, and type III collagen according to the sequence of discovery. Collagen is usually composed of 3 α-peptide chains, and the 3 peptide chains of some collagen molecules are the same, and some are different. According to international practice, the three peptide chains of collagen molecules are called α1-chain, α2-chain, and α3-chain. If two collagens are superimposed on each other, the name is accompanied by uppercase Roman numerals and enclosed in brackets, for example, the α1-chain of type III collagen is ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 鲁艳芹韩金祥张磊亮王延宙任秀智彭传明岳笑然
Owner SHANDONG FIRST MEDICAL UNIV & SHANDONG ACADEMY OF MEDICAL SCI
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