Method for detecting gastric cancer by detecting VLDLR gene

A technology of tissue cells and detection methods, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as difficulty in finding target genes, difficulties, time-consuming and laborious identification of specific gene deletions, etc.

Inactive Publication Date: 2007-12-26
FUJIFILM CORP +1
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  • Abstract
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  • Claims
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Problems solved by technology

In the case of using common methods such as positional cloning, the identification of specific gene deletions is very time-consuming and laborious, and it is difficult to find the target gene
Moreover, even with the CGH method, it is very difficult to detect deletions of genomic DNA less than 5-10 Mb in size by the usual metaphase chromosome method (Non-Patent Documents 2 and 3)

Method used

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  • Method for detecting gastric cancer by detecting VLDLR gene
  • Method for detecting gastric cancer by detecting VLDLR gene

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preparation example Construction

[0038] Dry spots can be prepared by dropping endless BAC DNA or the like on a substrate using a spotter, forming multiple spots, and then drying the spots. As the spotting device, an inkjet printer, a dot matrix printer, and a bubble jet (registered trademark) printer can be used, and an inkjet printer is preferably used. For example, GENESHOT (NGKINSULATORS Co., Ltd., Nagoya) etc. can be used.

[0039] In this way, a desired DNA-immobilization substrate can be prepared by immobilizing infinite BAC DNA or the like on a substrate, particularly preferably a solid substrate.

[0040] Further, as a method for directly detecting the deletion of the human VLDLR gene, the Northern blotting method can also be mentioned. The Southern blotting method is to separate and fix the genomic DNA obtained from the sample, and detect whether the gene exists in the sample by detecting its hybridization with the human VLDLR gene. In addition, both the base sequence and amino acid sequence of the...

Embodiment 1

[0056] From the genome database sites of National Cancer for Biotechnology and University of California Santa Crus Biotechnology and the BLAST search results of the selected DNA, genes or sequence tagged site markers (Sequence Tagged Site markers) that are extremely important for cancerization and cancer cell proliferation were selected. 800 BAC / PAC clones.

[0057] The BAC / PAC DNA was digested with DpnI, RsaI, and HaeIII, and ligated with adapter DNA (adaptor DNA). Next, 2 PCRs were performed using primers having linker sequences. One of the 5' ends of the two primers was aminated. This step is called "infinitization", and the resulting DNA is defined as "infinitization DNA". The infinite DNA was covalently printed on an oligomeric DNA microarray (Matsunami Glass Industry Co., Ltd., Osaka) twice using an inkjet spotter (GENESHOT, NGK Insulators, Nagoya).

Embodiment 2

[0059] In order to detect novel homozygous deletions in gastric cancer, CGH array analysis was performed using the CGH array of Example 1 using genomic DNA prepared from 32 types of gastric cancer cells.

[0060] In addition, as a control, genomic DNA from healthy males was used and labeled with Cy5. As DNA to be detected, genomic DNA prepared from the above gastric cancer cells was used and labeled with Cy3. Specifically, DpnI-digested genomic DNA (0.5 μg) in the presence of 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dGTP, 0.1 mM dCTP, and 0.4 mM Cy3-dCTP (gastric cancer cells) or 0.4 mM Cy5-dCTP (normal cells) Next, tagging is performed by nick translation. Cy3 and Cy5 labeled dCTPs were obtained from Amersham Biosciences (Tokyo). The two labeled genomic DNAs were added to ethanol in the presence of Cot-1 DNA (Invitrogen Co.), precipitated, and dissolved in 120 μl hybridization mixture (50% formamide, 10% dextran sulfate, 2×SSC ( 1×SSC: 150 mM sodium chloride / 15 mM sodium citrate))...

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Abstract

The method for detecting gastric cancer comprises detecting canceration of gastric tissue cell by detecting the inactivation of the human VLDLR (VLDL receptor) gene in the gastric tissue cell by (homozygote) deletion, methylation of CpG island, etc. A DNA chip method, a Southern blot method, a Northern blot method, a real time RT-PCR method, an FISH method, a CGH method, etc., may be cited as the detecting means.

Description

technical field [0001] The invention relates to a method for detecting the inactivation of human very low density lipoprotein receptor (Very LowDensity Lipoprotein Receptor) (human VLDLR) gene in gastric tissue cells. Background technique [0002] In Japan, more than 100,000 people suffer from gastric cancer (GC) every year, and the male-to-female ratio is about 2:1. It is the most common cancer among males. Gastric cancer kills about 50,000 people every year in Japan, accounting for 16% of all cancer deaths. According to previous studies, it is considered that gastric tissue cells undergo a series of genetic changes during the process of cell differentiation and proliferation, and as a result, canceration eventually occurs. However, it is still unclear exactly what genetic changes induce cancerous gastric tissue cells. Therefore, the current situation is that there is no detection method for gastric cancer and a detection method for the degree of malignancy of gastric can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q2600/154C12Q1/6886
Inventor 高田久井本逸势稻泽让治
Owner FUJIFILM CORP
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