title: Detecting low-abundance p16 and p53 mutations in pancreatic juice using a novel assay: heteroduplex analysis of limiting dilution PCRs.
abstract: We have developed a simple, robust, highly-sensitive assay for identifying gene mutations in clinical samples. We applied this assay to detect p53 and p16 mutations in pancreatic juice obtained from patients undergoing evaluation and treatment of pancreatic disease. The assay strategy involves PCR amplifying DNA at limiting dilution (LD-PCR) followed by screening PCR products for mutations using temperature gradient capillary electrophoresis (TGCE). Compared to conventional TGCE, TGCE after LD-PCR significantly increased the number of detectable mutations in pancreatic duct juice. Using LD-PCR, mutations in p53 and/or p16 were found in the pancreatic juice of 12 of 20 individuals with pancreatic cancer compared to only 1 of 8 patients with chronic pancreatitis, 0 of 8 individuals without evidence of pancreatic disease (p<0.02). We conclude that limiting dilution PCR is an effective strategy for improving the detection of mutations in clinical samples and when applied to pancreatic juice to detect mutations of p53 and/or p16, it can help distinguish patients with pancreatic cancer from those without evidence of pancreatic neoplasia.
authors.affiliation: Department of Pathology, The Sol Goldman Center for Pancreatic Cancer Research, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA.
authors.forename: Yansong
authors.initials: Y
authors.lastname: Bian
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Matsubayashi
authors.forename: Chung-Pin
authors.initials: CP
authors.lastname: Li
authors.forename: Tadayoshi
authors.initials: T
authors.lastname: Abe
authors.forename: Marcia
authors.initials: M
authors.lastname: Canto
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Murphy
authors.forename: Michael
authors.initials: M
authors.lastname: Goggins
mesh_terms: D003937:Diagnosis, Differential; D005091:Exons; D005784:Gene Amplification; D019942:Genes, p16; D016158:Genes, p53; D006801:Humans; D009154:Mutation; D010182:Pancreatic Diseases / Q000175:diagnosis / Q000235:genetics*; D010189:Pancreatic Juice / Q000502:physiology*; D010190:Pancreatic Neoplasms / Q000175:diagnosis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods