Development of novel screening method by detection of APC mutation from colorectal cancer cells in stool

我们注意到80-90%的结直肠癌存在APC（adenomatous polyposis coli）基因突变。我们的目的是检测癌症的存在，提取粪便中脱落的癌细胞的DNA，并在本项目中使用酵母菌.人结直肠癌细胞系：SW 480在APC基因的MCR（突变簇病变）中存在导致终止密码子的突变。我们从正常淋巴细胞DNA和突变DNA中扩增APC基因的MCR损伤，并将其导入URA 3（+）载体后进行酵母转化。然后在正常和5 FOA（+）培养基中测定酵母菌的菌落形成情况。由于APC的终止密码子突变导致URA 3缺失，在包含来自SW 480的APC的酵母中检测到集落形成，而在包含来自正常DNA 2的APC的酵母中未观察到集落。其次，我们在已建立的粪便DNA提取技术的基础上，对其用量和方法作了进一步的改进。成功获得稳定的DNA提取物.建立了从正常人粪便中提取DNA、APC MCR、酵母转化的技术。然而，仍需要进一步提高DNA转化的效率。对于癌症患者，已经完成了从样品中提取APC的DNA序列的技术。现在我们正在计划一个基因分析咨询医院伦理委员会。现在我们还没有申请专利，因为这项研究还不够成熟。我们正在计划进一步审查申请。

We noticed that 80-90% of colorectal cancer have APC (adenomatous polyposis coli) gene mutations. We aimed to detect the presence of cancer extracting DNA of fallen cancer cells in stool and using yeast in this project.1. Cell line from human colorectal cancer : SW480 has mutation that causes stop codon in MCR (mutation cluster lesion) of APC gene. We amplified MCR lesion of APC gene of normal DNA from lymphocyte and mutated DNA from SW480 and caused yeast transformation after transducing into URA3(+) vector. Then we tested colony formation of yeast in normal and 5FOA(+) culture medium. Colony formation was detected in yeast including APC from SW480 due to stop codon of APC mutation coursing absence of URA3, while colony was not seen in yeast including APC from normal DNA2. Next, we added further improvement concerning its amount and method on established technique of DNA extraction from stool. Stable DNA extraction was successfully obtained.3. We have established the technique of DNA extraction from stool of normal volunteer, per of APC MCR, transformation of yeast. However, further improvement of efficiency of DNA transformation is still needed. For cancer patient, technique of DNA sequence of APC from samples has been completed. Now we are planning a gene analysis consulting hospital ethics committee4. Now we have not applied a patent because this study is not mature enough. We are planning the application with further examination.