Dection of mutations in cancer-related genes using Muts mismatch binging protein and its clinical application to diagnosis of digestive cancers.

最近，在Lishanski等人和Wagner等人的实验报告中，展示了利用Mut S错配结合蛋白检测遗传异常的高灵敏度方法，包括所有单碱基突变，以及任何位置的小缺失或插入。我试图将这种新方法应用于检测癌症相关基因的变化。首先，从5例胰腺癌（PCa）和10例慢性胰腺炎（CP）患者的4株胰腺癌细胞系和胰液（PJ）中提取DNA。合成了K-ras密码子12、p53外显子5 ~ 8和p16外显子1 ~ 3的生物素标记义引物。经过触地PCR扩增、热变性和再退火过程后，在存在错配和未配对碱基的情况下形成异双工PCR产物，否则形成单双工PCR产物。由于异双相而非同双相与固定在硝化纤维素膜上的MutS蛋白具有特异性结合，本文采用链霉亲和素- hrp的化学发光方法对与MutS蛋白特异性结合的异双相进行了检测。在4种胰腺癌细胞系PANC-1、PaCa-2、BxPC-3和HPAF中，理论上，PaCa-2 （K-ras密码子12纯合突变）和BxPC-3 （K-ras密码子12无突变）单独形成同工双工。PANC-1和HPAF也会由于K-ras密码子12的杂合突变而形成异双工。另一方面，通过直接测序已经在PANC-1（外显子8）、PaCa-2（外显子7）、BxPC-3（外显子6）、HPAF（外显子5）中检测到p53突变。因此，以同样的方式，这些p53突变在理论上形成异双工。然而，K-ras外显子1和p53外显子5在PaCa-2、p53外显子6在PaCa-2和p53外显子7在BxPC-3中被识别为假阳性。此外，尽管使用MutS法检测K-ras外显子1的异双工发生率为80% (4/5)，p53在PCa的PJ中为60% (3/5)，K-ras外显子1的异双工发生率为60% (6/10)，p53在cp的PJ中为50%(5/10)，这种检测方法灵敏度高，但存在假阳性频繁的问题。假阳性的原因被认为是在PCR过程中掺入错误，以及与固定化MutS结合蛋白反应后，硝化纤维素膜上的非特异性生物去除不完全。这种检测方法还需要进一步改进。少

Recently, highly sensitive method of detecting genetic abnormalities including all single base mutations, as well as small deletions or insertions at any locations using Mut S mismatch binding protein was demonstrated in the experimental reports of Lishanski et al. and Wagner et al. I tried to apply this new method to detection of changes of cancer-related genes. First, DNA was extracted from 4 pancreatic cancer cell lines and pancreatic juice (PJ) from 5 patients with pancreatic carcinoma (PCa) and 10 with chronic pancreatitis (CP). Biotin-labeled sense primers for K-ras codon 12, exon 5 to 8 of p53, and exon 1 to 3 of p16 were synthesized. After the process of touchdown PCR amplification, heat denaturation and re-annealing, heteroduplex PCR products form in the case of presence of mispaired and unpaired bases and otherwise homoduplex PCR products alone form. Because of the specific binding of the heteroduplex not homoduplex to immobilized MutS protein on the nitrocellulose membrane, … More chemiluminescence method using streptavidin-HRP was performed for the detection of heteroduplex which is specific binding to MutS protein.Among 4 pancreatic cancer cell lines, that is, PANC-1, PaCa-2, BxPC-3, and HPAF, PaCa-2 (homozygous mutation of K-ras codont 12) and BxPC-3 (no mutation of K-ras codon 12) would theoretically form homoduplex alone. PANC-1 and HPAF would also form heteroduplex due to heterozygous mutation of K-ras codon 12. On the other hand, p53 mutations were already detected in PANC-1 (exon 8), PaCa-2 (exon 7), BxPC-3 (exon 6), HPAF (exon 5) by direct sequencing. Therefore, as the same manner, these p53 mutations theoretically form heteroduplex. However, false positive was recognized in PaCa-2 for K-ras exon 1 and in PANC-1 for p53 exon 5, PaCa-2 for p53 exon 6, and BxPC-3 for p53 exon 7.Moreover, although the incidence of this heteroduplex using MutS assay was 80% (4/5) for K-ras exon 1 and 60% (3/5) for p53 in PJ with PCa, it was also 60% (6/10) for K-ras exon 1 and 50% (5/10) for p53 in PJ with CP.This detecting method has high sensitivity, but the problem of frequent false positivity.The cause of the false positivity was regarded as the misincorporation during PCR and imperfect removal of non-specific biotion from the nitrocellulose membrane after the reaction with immobilized MutS binding protein. Further improvement will be needed for this detecting method. Less