Epithelial type cadherin expressed gene CDH1 mutation detection kit and application thereof

A detection kit, kit technology, applied in the field of genetic diagnosis and consequence assessment

Inactive Publication Date: 2011-02-16
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The functional consequences of detected missense mutations and promoter region mutations have not been reported

Method used

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  • Epithelial type cadherin expressed gene CDH1 mutation detection kit and application thereof
  • Epithelial type cadherin expressed gene CDH1 mutation detection kit and application thereof
  • Epithelial type cadherin expressed gene CDH1 mutation detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Systematic screening and case-control analysis of CDH1 gene mutation in Chinese patients with gastric cancer

[0033] A total of 238 patients with gastric cancer in Jiangsu, Anhui and Zhejiang regions of China and 240 normal controls were selected for peripheral blood extraction and DNA extraction. For exon 2-12 and 14-16 of CDH1 gene, primers were designed according to Table 1 for PCR amplification and high-resolution melting curve (HRM) analysis. The reaction system is: Genomic DNA (100ng / μl) 1.0μl, dNTP Mixture (2.5mM each) 0.8μl, 10×PCR buffer (Mg 2+ free) 1.0μl, MgCl 2 (25mM) 1.0μl, upstream primer (10μM) 0.2μl, downstream primer (10μM) 0.2μl, dimethyl sulfoxide (DMSO) 0.4μl, ddH 2 O 4.32 μl, LC Green dye 1.0 μl, Taq DNA polymerase (5 Unit / μl) 0.08 μl. Reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, renaturation at 60°C (corresponding annealing temperature) for 30 sec, extension at 72°C for 40 sec, 40 cycles; ...

Embodiment 2

[0039] Example 2: Analysis of the impact of detected CDH1 gene missense mutations on protein function

[0040] Using SIFT analysis (the Sorting Intolerant from Tolerant, http: / / sift.jcvi.org / ) and PolyPhen (=Polymorphism Phenotyping, http: / / genetics.bwh.harvard.edu / pph / ), analysis of missense mutations caused Effects of single amino acid changes on CDH1 protein function. The score threshold given by SIFT is 0.5, which means that those 0.5 are tolerable. The PolyPhen tool calculates the difference between the PSIC score of the encoded amino acid before and after the mutation to determine the impact of the mutation on the structure and function of the protein. When the difference exceeds 1.5, it is considered a mutation that changes the function. SIFT analysis in this study showed that the detected CDH1 c.1888C>G(L630V) mutation scored 0.02, while the PolyPhen score difference reached 1.748, so this mutation may affect the function of CDH1 protein. But V202I and T340A are only...

Embodiment 3

[0043] Example 3: Functional Consequence Analysis of Detected CDH1 Gene Promoter Region Mutations

[0044] The functional consequences of mutations in gene promoter regions were detected using a dual-luciferase reporter gene assay. Various mutant plasmids in the promoter region of PGL3-CDH1 were constructed, transfected into Hela cells, and the promoter activity intensity of each mutant gene was evaluated according to the relative luciferase activity of the expressed products.

[0045] (1) Construction of various PGL3-CDH1 promoter region mutant plasmids:

[0046] According to the sequence of CDH1 gene promoter region, design primers with double restriction sites. PCR amplification included a region from -345 to +271bp from the transcription initiation site. The upstream primer is PF: 5'-ATGC CTCGAG CCATTCTCCAAAACGAACAAAC-3' (SEQ ID NO: 39), 'ATGC' is a protection sequence,' CTCGAG ' is the recognition sequence of XhoI endonuclease. The downstream primer is PR: 5'-ATGC ...

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Abstract

The invention relates to a CDH1 mutation detection kit and an application thereof. The kit mainly comprises the following main ingredients: (1) 19 pairs of CDH1 gene PCR amplifying and sequencing primers; and (2) PCR reagent for amplification. The PCR amplification product obtained by the kit can screen gene micromutation by a high-resolution dissolved curve process or carries out polymorphism analysis on restrictive segment length. The kit has high-efficiency mutation detection and functional evaluation actions. The invention is used for CDH1 gene mutation detection.

Description

technical field [0001] The invention relates to a CDH1 gene mutation detection kit, in particular to its gene diagnosis and consequence evaluation in East Asian gastric cancer high-incidence population. Background technique [0002] The CDH1 gene is located on human chromosome 16q22.1, contains 16 exons, and is about 100kb in length. Its transcript is 4.5kb messenger RNA, which synthesizes 120kD E-cadherin (E-cad) through translation. E-cad is a member of the cadherin family, and the cadherin family molecules are transmembrane glycoproteins that mediate calcium-dependent intercellular adhesion. E-cad plays a key role in epithelial cell differentiation and maintenance of cell polarity. Abnormal expression of E-cad due to CDH1 gene mutation plays an important role in the formation of gastric cancer and other tumors. Therefore, CDH1 gene is an important susceptibility gene for gastric cancer. However, due to the limitations of detection methods, there were few systematic repo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 范怡梅陈琴花邓伟周函郭文文王亚平
Owner NANJING UNIV
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