Banerjee S K, Makdisi W F, Weston A P, Campbell D R
Molecular Gastroenterology and Pancreatic Cancer Research Unit, V.A. Medical Center, Kansas City, Missouri, USA.
Pancreas. 1997 Jul;15(1):16-24. doi: 10.1097/00006676-199707000-00003.
Mutations at codon 12 of the K-ras gene have been detected in pancreatic adenocarcinomas by a variety of techniques. A few of these techniques are very sensitive, identifying the mutations in 96-100% of cases. However, these sensitive techniques are labor intensive, utilizing multistep processing and radioactive material. Much simpler techniques, involving nonradioactive single-step polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) have been employed to detect K-ras mutations at codon 12 in pancreatic adenocarcinomas. However, the low sensitivity of these single-step PCR/ RFLP techniques is unacceptable. A simple and nonradio-active PCR/RFLP-based method for detection of K-ras codon 12 mutations in formalin-fixed, paraffin-embedded tissue sections of pancreatic adenocarcinoma is described and compared to the traditional PCR technique. K-ras gene mutations at codon 12 were detected by a modified two-step enrich-nested PCR (EN-PCR)/RFLP method, and their existence was confirmed by direct DNA sequencing analysis of the product. When the two-step EN-PCR/RFLP technique was compared to the single-step PCR/RFLP method, K-ras codon 12 mutations were detected in 100% of pancreatic adenocarcinomas (15/15) with the EN-PCR/RFLP method, while half as many (9/15) were detected with the single-step PCR/RFLP method. This study demonstrates that the sensitivity of the simple two-step EN-PCR/RFLP technique is comparable to that of the more complex methods for detecting K-ras mutations at codon 12 in formalin-fixed, paraffin-embedded tissue sections of pancreatic adenocarcinoma and its sensitivity is superior to that of the single-step technique.
通过多种技术已在胰腺腺癌中检测到K-ras基因第12密码子的突变。其中一些技术非常灵敏，能在96%-100%的病例中识别出这些突变。然而，这些灵敏技术劳动强度大，需要多步骤处理并使用放射性材料。更简单的技术，包括非放射性单步聚合酶链反应（PCR）-限制性片段长度多态性（RFLP）已被用于检测胰腺腺癌中K-ras基因第12密码子的突变。然而，这些单步PCR/RFLP技术的低灵敏度是不可接受的。本文描述了一种基于简单且非放射性PCR/RFLP的方法，用于检测福尔马林固定、石蜡包埋的胰腺腺癌组织切片中K-ras基因第12密码子的突变，并将其与传统PCR技术进行比较。通过改良的两步富集巢式PCR（EN-PCR）/RFLP方法检测K-ras基因第12密码子的突变，并通过对产物进行直接DNA测序分析来确认其存在。当将两步EN-PCR/RFLP技术与单步PCR/RFLP方法进行比较时，EN-PCR/RFLP方法在100%（15/15）的胰腺腺癌中检测到K-ras基因第12密码子的突变，而单步PCR/RFLP方法仅检测到一半数量（9/15）的突变。本研究表明，简单的两步EN-PCR/RFLP技术在检测福尔马林固定、石蜡包埋的胰腺腺癌组织切片中K-ras基因第12密码子突变的灵敏度与更复杂的方法相当，且其灵敏度优于单步技术。
