Image analysis in quantitative PCR. An application for the measurement of c-erbB-2 oncogene amplification in DNA from human tumours.
We present an application of image analysis for the direct quantification of PCR products after gel electrophoresis and ethidium bromide staining of DNA. This procedure has been applied to the development of an assay based on competitive PCR for the measurement of the degree of amplification of c-erbB-2 oncogene in DNA from human tumours. In this method two DNA species (genomic and competitor) compete for PCR amplification. Since results are calculated from the final competitor/genomic ratio any variable affecting the rate of PCR amplification has no effect on the accuracy of the ratio measurement. Results are reported which show that even large variations in the experimental conditions (number of PCR cycles, sample volumes and extracted DNA quality) did not interfere with the precision of the measurement of the competitor/genomic ratio.
Orlando
C
C
Department of Clinical Physiopathology, University of Florence, Italy.
Sestini
R
R
Zentilin
L
L
Gelmini
S
S
Pinzani
P
P
Giacca
M
M
Pazzagli
M
M
D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis / Q000378:metabolism*; D005260:Female; D006801:Humans; D008163:Luminescent Measurements; D009369:Neoplasms / Q000235:genetics* / Q000378:metabolism; D010783:Photometry / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D011519:Proto-Oncogenes; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics
D004273:DNA, Neoplasm; D018719:Erb-b2 Receptor Tyrosine Kinases