[Detection of APC mutation by real-time polymerase chain reaction and melting curve analysis]
Wei-wei Zheng1, Zhong-chen Liu, Qing-ge Li
1Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of Education, Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, China.
Objective:
To establish a simple, reliable and low cost approach for clinical detection of APC mutation.
Methods:
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.
Results:
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.
Conclusions:
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.
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