[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.
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.
Wei-wei
WW
Zheng
Zhong-chen
ZC
Liu
Qing-ge
QG
Li
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