Measuring c-erbB-2 oncogene amplification in fresh and paraffin-embedded tumors by competitive polymerase chain reaction.
We present an original application of competitive polymerase chain reaction (PCR) for measuring oncogene amplification in DNA from human tumors by simultaneous PCR amplification of genomic DNA with fixed amounts of an internal standard (competitor DNA). Competitors share the same sequence as the target genes but contain an additional 15- to 20-base-pair insert, which allows resolution of the amplified products after polyacrylamide gel electrophoresis and ethidium bromide staining. The gene copy number is derived from the ratio between the intensities of the bands corresponding to the amplified products. Using this procedure, we measured c-erbB-2 amplification in breast and bladder carcinomas in both fresh tumor tissues and paraffin-embedded tissue samples and assessed the precision, sensitivity, and accuracy of the assay. Competitive PCR is a simple, reliable, and accurate method for the evaluation of c-erbB-2 amplification and is potentially suitable for use in the clinical laboratory.
Sestini
R
R
Department of Clinical Physiopathology, University of Florence, Italy.
Orlando
C
C
Zentilin
L
L
Gelmini
S
S
Pinzani
P
P
Bianchi
S
S
Selli
C
C
Giacca
M
M
Pazzagli
M
M
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000235:genetics*; D004996:Ethidium; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D010232:Paraffin; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D018719:Erb-b2 Receptor Tyrosine Kinases; D013194:Staining and Labeling; D016610:Tissue Embedding; D001749:Urinary Bladder Neoplasms / Q000235:genetics*
D004273:DNA, Neoplasm; D066246:ErbB Receptors; D004996:Ethidium; D010232:Paraffin; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases