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Primer, method and kit for detecting whole exon mutation of SMAD4 gene

An all-exon and exon technology, which is used in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection to ensure accuracy and uniqueness, improve risk assessment, and simplify the method.

Pending Publication Date: 2022-05-13
合肥艾迪康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] Although the specific regulatory mechanism of SMAD4 at many levels, complex and coordinated, and the relationship between its regulatory imbalance and tumors have not been reported, in recent years, the evaluation and prediction of SMAD4 gene mutations in the metastasis and prognosis of gastrointestinal diseases have become more and more important. Many, through the detection of the SMAD4 gene in patients, it can assist in the diagnosis of diseases, prevent some cancers, reduce the risk of cancer, and choose a more reasonable and effective treatment plan. Therefore, it is very important to perform whole exome sequencing on the SMAD4 gene. significance

Method used

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  • Primer, method and kit for detecting whole exon mutation of SMAD4 gene
  • Primer, method and kit for detecting whole exon mutation of SMAD4 gene
  • Primer, method and kit for detecting whole exon mutation of SMAD4 gene

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Experimental program
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Effect test

Embodiment 1

[0088] The primer sequence for detecting SMAD4 gene full-exon mutation includes forward and reverse primers that amplify and cover SMAD4 full-exon mutation site, and its base sequence is:

[0089] SMAD4-1F: CAGGTGTGTCGTAGGATTCG

[0090] SMAD4-1R: GGGAGAAGGTGGCTAGGTTG

[0091] SMAD4-2F: TTTCCTTGCAACGTTAGCTG

[0092] SMAD4-2R: GGAGCACAAATTAAATTACCCTG

[0093] SMAD4-3F: CTGAGTTGGTAGGATTGTGAGG

[0094] SMAD4-3R: TGAAACACTATTGAGATCCTTTTCC

[0095] SMAD4-4F: GCGTTTATGCTACTTCTGAATTG

[0096] SMAD4-4R: TTAATGTTACTGCCTGCCGC

[0097] SMAD4-5F: CCGCTGAATAAATGACTTTTGC

[0098] SMAD4-5R: TTCCAAGTGATTGTGCATACC

[0099] SMAD4-6-7F: CCCATCTTTTATAGTTGTGCATTATC

[0100] SMAD4-6-7R: AAAACAGAAAACAAAGCCCTACC

[0101] SMAD4-8F: TTGGCAGATAGCACTGAAATG

[0102] SMAD4-8R: CCCCATAATTCCATTAAAGCC

[0103] SMAD4-9F: TGTGGAGTGCAAGTGAAAGC

[0104] SMAD4-9R: TGTACATGGGAAAACATAACCTTG

[0105] SMAD4-10F: GAATTCATACTACATGCTCCTGACAC

[0106] SMAD4-10R:TTTTCCATTCCTTCCACCCC

[0107] SMAD4-11F: TCCAAG...

Embodiment 2

[0116] The kit for detecting SMAD4 gene mutations includes: a detection system PCR amplification reaction solution and a sequencing system reaction solution, wherein the detection system PCR amplification reaction solution includes: 2×PCR Buffer; dNTPs (2mM); KOD FX DNA Polymerase (1U / μl); the upper and lower primers SMAD4-1F (10 μM), SMAD4-1R (10 μM), SMAD4-2F (10 μM), SMAD4-2R (10 μM), SMAD4-3F (10 μM), SMAD4-3R(10μM), SMAD4-4F(10μM), SMAD4-4R(10μM), SMAD4-5F(10μM), SMAD4-5R(10μM), SMAD4-6-7F(10μM), SMAD4-6-7R( 10μM), SMAD4-8F(10μM), SMAD4-8R(10μM), SMAD4-9F(10μM), SMAD4-9R(10μM), SMAD4-10F(10μM), SMAD4-10R(10μM), SMAD4-11F(10μM ), SMAD4-11R (10 μM), SMAD4-12F (10 μM), SMAD4-12R (10 μM).

[0117] The sequencing system reaction solution includes: sequencing purification solution (exonuclease I: 0.6U, calf small intestine alkaline phosphatase: 1.2U); EDTA (125mM); absolute ethanol; 75% ethanol; HIDI (highly deionized formamide ); a pair of sequencing primers M13F (3.2 μM), ...

Embodiment 3

[0121] Method for detecting SMAD4 gene mutation site

[0122] (1) DNA extraction from tissue samples

[0123] Extract DNA from human tissue samples (using QIAamp DNA FFPE TissueKit, a DNA extraction kit from Qiagen), the specific extraction method is as follows:

[0124] 1) Remove the non-cancerous tissue with a razor blade, collect the paraffin-embedded tissue into a 1.5mL centrifuge tube, and centrifuge briefly.

[0125] 2) Add 1mL tissue clear solution, shake fully, centrifuge at 13200rpm for 1min, and discard the supernatant.

[0126] 3) Add 0.5mL tissue clear solution, shake fully, centrifuge at 13200rpm for 1min, and discard the supernatant.

[0127] 4) Add 1 mL of absolute ethanol, shake vigorously, centrifuge at 13200 rpm for 2 min, and discard the supernatant.

[0128] 5) Open the lid and incubate at room temperature or in a metal bath at 37°C until the ethanol evaporates completely.

[0129] 6) Take 180 μL Buffer ATL to resuspend the tissue in the tube, add 20 μL...

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Abstract

The invention discloses a primer, a method and a kit for detecting whole exon mutation of an SMAD4 gene. The specific primer comprises forward and reverse primers for amplifying and covering all 12 exons of the SMAD4 gene; according to the invention, the Sanger sequencing method is adopted to detect the mutation of the whole exons of the SMAD4 gene, and the mutation of the whole exons of the SMAD4 gene can be rapidly detected. The detection result completed by the method is accurate, whether the SMAD4 gene is mutated or not can be specifically identified, and the accuracy and uniqueness of the determination result are ensured; the kit can assist in disease diagnosis and improve risk assessment of diseases, and has important reference significance for early intervention and early treatment.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a primer, a method and a kit for detecting SMAD4 gene whole exon mutation. Background technique [0002] SMAD4 gene is a new type of tumor suppressor gene, which was first discovered in pancreatic cancer by Hahn et al. It is also called pancreatic cancer deletion gene 4 (Deleted in Pancreatic Cancer-4, DPC4), located on human chromosome 18q21.1, the gene sequence Containing 12 exons, the transcript length is 2680bp, and the encoded protein SMAD4 consists of 552 amino acid residues. The protein encoded by SMAD4 belongs to the SMAD family, can be activated by transmembrane serine: threonine receptor kinase, and is a signal transduction protein. Most studies believe that SMAD4 acts as a key intermediate conduction molecule in the TGF-β signal transduction pathway, and all biological effects are the result of the interaction between the receptor regulatory protein...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/156C12Q2600/166C12Q2531/113C12Q2545/113C12Q2535/101
Inventor 蔡嘉慧王淑一
Owner 合肥艾迪康医学检验实验室有限公司
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