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Articles |
1
Perkin-Elmer Italy, Monza, Milan, Italy.
a Author for correspondence. Fax +39.55.4377290.
We describe a PCR-based assay for determining
c-erbB-2 oncogene amplification in breast
cancer in which we use the TaqManTM system. Two fluorogenic
probes anneal to the target between primers for
c-erbB-2 and ß-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.
Key Words: indexing terms: breast cancer • genetic alterations • oligonucleotide probes • fluorescence assays
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