Detecting low-abundance p16 and p53 mutations in pancreatic juice using a novel assay: Heteroduplex analysis of limiting dilution PCRs

We have developed a simple, robust, and 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 (LDPCR) followed by screening PCR products for mutations using temperature gradient capillary electrophoresis (TGCE). Compared to conventional TGCE, TGCE after LDPCR 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, and 0 of 8 individuals without evidence of pancreatic disease (p