Detection of APC mutation by real-time polymerase chain reaction and melting curve analysis

Abstract

To establish a simple, reliable and low cost approach for clinical detection of APC mutation. Using SYBR Green I as the real-time polymerase chain reaction (PCR) product indicator, a DNA fragment of 270 bp targeting APC_1309 mutation (5 bp deletion) was amplified from the sample DNA. A short fragment (40/35 bp) was then amplified from the 270 bp PCR product, followed by melting curve analysis from 65 degrees C to 99 degrees C at 0.5 degrees C/step. A total of 18 paraffin-embedded tumor samples were analyzed, of which 7 were tested positive for the mutation and 11 were negative. No mutation was detected in any of the 20 normal peripheral blood samples. Real-time PCR melting curve analysis can be used for routine APC mutation detection. The simple design, low cost and high reliability should allow similar applications to the analysis of a variety of other gene mutations.