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

我们注意到80-90%的结直肠癌有APC（大肠腺瘤性息肉病）基因突变。我们的目的是检测癌症的存在，提取粪便中落下的癌细胞的DNA，并使用酵母。人结直肠癌细胞系SW480突变导致APC基因MCR（突变簇病变）停止密码子。我们从淋巴细胞中扩增正常DNA的APC基因MCR病变，从SW480中扩增突变DNA的APC基因MCR病变，并将其转导到URA3（+）载体中引起酵母转化。然后，我们测试了酵母在正常和5FOA（+）培养基中的菌落形成。由于APC突变的终止密码子缺失URA3，在SW480的APC酵母中检测到集落形成，而在正常DNA2的APC酵母中未见集落形成。其次，在已建立的粪便DNA提取技术基础上，对其提取量和提取方法进行了进一步改进。成功地获得了稳定的DNA提取。我们建立了正常志愿者粪便DNA提取技术，根据APC MCR，酵母转化。然而，DNA转化的效率还有待进一步提高。对于癌症患者，已经完成了样本APC DNA序列测定技术。现在我们正在计划进行基因分析，咨询医院伦理委员会。目前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的审查申请。

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.