Janda J, Krechler T, Dufek V, Klener P
I. interní klinika 1. LF UK a VFN, Praha.
Cesk Patol. 1998 Jan;34(1):33-7.
Present study was undertaken to detect Ki-ras point mutation at codon 12 in pancreatic adenocarcinomas (CaP) using the polymerase chain reaction-restriction fragment lengths polymorphism (PCR-RFLP). Three modifications of PCR-RFLP were performed with a mismatched primers creating a recognition site with only one allelic from (wild or mutated). Using two-step PCR-RFLP and two modifications of one-step PCR-RFLP we examined 5 resected adenocarcinomas of pancreas, 6 pancreatic juices and one DNA sample from peripheral blood of patient with generalized stadium of CaP. We compare all techniques and conclude, that the very sensitive two step PCR-RFLP is a suitable method for detection point mutations and eliminates the need for either oligonucleotide hybridization or DNA sequencing.
本研究旨在利用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)检测胰腺腺癌(CaP)中第12密码子的Ki-ras点突变。采用错配引物进行了三种PCR-RFLP改良方法,仅从一个等位基因(野生型或突变型)创建识别位点。我们使用两步PCR-RFLP以及一步PCR-RFLP的两种改良方法,检测了5例胰腺切除腺癌、6份胰液以及1例处于CaP广泛期患者外周血的DNA样本。我们比较了所有技术并得出结论,非常灵敏的两步PCR-RFLP是检测点突变合适的方法,无需进行寡核苷酸杂交或DNA测序。