Analysis of gene amplification in archival tissue by differential polymerase chain reaction.
Oncogene amplification is found in many human tumors, and its detection may have important prognostic value. However, analysis of gene amplification may be hampered by inadequate tissue or poor DNA quality. We have previously described a polymerase chain reaction (PCR)-based procedure called differential PCR that can detect variations in gene dosage using miniscule amounts of tumor DNA [Frye, R.A., Benz, C.C. & Liu, E. (1989). Oncogene, 4, 1153-1157]. We now report the optimization of this technique for the analysis of oncogene amplification in paraffin-embedded archival tissues. We find that differential PCR is able to detect amplification of the HER2 (c-erbB-2) and the epidermal growth factor receptor (EGFR) genes and can be used to arrive at a semiquantitative estimate of gene dosage. Furthermore, our approach can determine gene amplification in samples in which the DNA is significantly degraded. Using differential PCR on paraffin-embedded tissues from cases previously investigated by standard DNA extraction and dot-blot procedures, good correlation between the two methods was found. Approaches are described to overcome technical problems posed by factors that affect the differential PCR, including the method of DNA extraction and extreme fragmentation of the DNA (less than 200 base pairs). Furthermore, the resulting analytical algorithm reported herein has proved effective in detecting oncogene amplification in archival breast cancer specimens from standard pathology laboratories. Thus, differential PCR will be particularly helpful in the analysis of tumor specimens that are archived, small in size or rare in occurrence.
Department of Medicine and Genetics, Lineberger Cancer Research Center, University of North Carolina, Chapel Hill 27599-7295.
A
A
Neubauer
B
B
Neubauer
M
M
He
P
P
Effert
D
D
Iglehart
R A
RA
Frye
E
E
Liu
D000465:Algorithms; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics; D002460:Cell Line; D004247:DNA / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D015345:Oligonucleotide Probes; D010232:Paraffin; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D018719:Erb-b2 Receptor Tyrosine Kinases; D014021:Tissue Preservation
D004247:DNA; D015345:Oligonucleotide Probes; D010232:Paraffin; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases