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等人的实验报告中，证明了使用Mut S错配结合蛋白检测包括所有单碱基突变以及任意位置的微小缺失或插入在内的遗传异常的高灵敏度方法。和Wagner等人。我尝试将这种新方法应用于检测癌症相关基因的变化。首先，从4个胰腺癌细胞系和5例胰腺癌(PCa)和10例慢性胰腺炎(CP)患者的胰液中提取DNA。合成了K-ras基因第12密码子、p53基因第5~8外显子和p16基因第1~3外显子的生物素标记正义引物。经降落式PCR扩增、热变性和复退火后，在有错配碱基和未配对碱基的情况下形成异源双链PCR产物，否则单独形成同源双链PCR产物。由于硝酸纤维素膜上固定的MutS蛋白与异源双链而不是同源双链具有特异性结合，…在PANC-1、PACA-2、BxPC-3和HPAF 4个胰腺癌细胞系中，PACA-2(K-ras密码子12纯合子突变)和BxPC-3(K-ras密码子12无突变)理论上单独形成同源双链。而PANC-1(外显子8)、PACA-2(外显子7)、BxPC-3(外显子6)、HPAF(外显子5)已检测到PANC-1(外显子8)、PACA-2(外显子7)、BxPC-3(外显子6)、HPAF(外显子5)中的p53突变。因此，以同样的方式，这些p53突变在理论上形成了异源双链。但在PJK-ras外显子1、PANC-1、P53外显子6、PacA-2、P53外显子7的PACA-2、PACA-2、P53外显子7的假阳性分别为PACA-2、PACA-2、P53外显子7的假阳性。MutS法检测PJ中K-ras外显子1的假阳性率为80%(4/5)、P53外显子7的假阳性率为60%(3/5)，而用MutS法检测PJ中K-ras外显子1的假阳性率为60%(6/10)、P53的假阳性率为50%(5/10)。造成假阳性的原因被认为是聚合酶链式反应过程中的错误掺入，以及与固定化MutS结合蛋白反应后硝酸纤维素膜上的非特异性生物去除不彻底所致。这种检测方法还需要进一步的改进。较少

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