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(腺瘤性息肉病)基因突变。在这个项目中，我们的目标是检测癌症的存在，提取粪便中脱落的癌细胞的DNA，并使用酵母。人结直肠癌细胞株：SW480在APC基因的MCR(突变簇损伤)中存在导致终止密码子突变。我们从淋巴细胞中扩增出正常DNA的APC基因的MCR损伤，并从SW480中突变出DNA，转化到URA3(+)载体后进行酵母转化。然后检测酵母在正常和5FOA(+)培养基中的集落形成情况。在酵母中，包括SW480的APC在内，由于APC突变的终止密码子导致URA3缺失而形成菌落，而在包括正常DNA2的APC在内的酵母中未见菌落形成。其次，在已建立的粪便DNA提取技术的基础上，对其用量和方法进行了进一步改进。成功获得了稳定的DNA提取。建立了正常志愿者粪便DNA的提取、APC-MCR的DNA提取、酵母转化的方法。但DNA转化效率仍需进一步提高。对于癌症患者，已经完成了从样本中提取APC的DNA测序技术。现在我们正在计划一个基因分析咨询医院伦理委员会4。现在我们还没有申请专利，因为这项研究还不够成熟。我们正在计划申请，并进行进一步的审查。

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.