Quantitative polymerase chain reaction-based homogeneous assay with fluorogenic probes to measure c-erbB-2 oncogene amplification.
We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqMan system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and beta-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5'-->3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P < 0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens.
Gelmini
S
S
Department of Clinical Physiopathology, University of Florence, Italy.
Orlando
C
C
Sestini
R
R
Vona
G
G
Pinzani
P
P
Ruocco
L
L
Pazzagli
M
M
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005914:Globins / Q000235:genetics; D006801:Humans; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*; D012235:Rhodamines; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
D004273:DNA, Neoplasm; D004259:DNA-Directed DNA Polymerase; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005914:Globins; D015345:Oligonucleotide Probes; D018719:Erb-b2 Receptor Tyrosine Kinases; D012235:Rhodamines; D019914:Taq Polymerase; C106141:6-carboxytetramethylrhodamine; C024098:6-carboxyfluorescein