[Detection of APC mutation by real-time polymerase chain reaction and melting curve analysis].
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.
Zheng
Wei-wei
WW
Key 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.
Liu
Zhong-chen
ZC
Li
Qing-ge
QG
D025601:Adenomatous Polyposis Coli Protein / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis; D017491:Genes, APC; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D017384:Sequence Deletion
D025601:Adenomatous Polyposis Coli Protein; D004273:DNA, Neoplasm