A two-step enriched-nested PCR technique enhances sensitivity for detection of codon 12 K-ras mutations in pancreatic adenocarcinoma.
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.
Banerjee
S K
SK
Molecular Gastroenterology and Pancreatic Cancer Research Unit, V.A. Medical Center, Kansas City, Missouri, USA.
Makdisi
W F
WF
Weston
A P
AP
Campbell
D R
DR
D001483:Base Sequence; D018270:Carcinoma, Ductal, Breast / Q000235:genetics* / Q000473:pathology; D003062:Codon / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000032:analysis; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000473:pathology; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D012680:Sensitivity and Specificity
D003062:Codon; D017931:DNA Primers; D004273:DNA, Neoplasm; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)