Oncogene amplification screening by labeled primer multiplex polymerase chain reaction.
This paper describes an improved procedure for rapid detection of amplified genes in fresh or formalin-fixed, paraffin-embedded tissues. Utilizing a multiplex differential polymerase chain reaction with radioactively labeled primers and electrophoresis of the products through thin gels, it is possible to screen for oncogene amplification more rapidly and reproducibly than has been previously demonstrated. This procedure takes advantage of thin vertical gels with external cooling, which allows sharp band resolution. Four separate gels can be electrophoresed at the same time in a single gel box. Because each gel slab contains 10 or more lanes, 40 or more samples can be assayed for gene amplification simultaneously. The entire procedure can be carried out from formalin-fixed, paraffin-embedded tissue to finish in 8 h when combined with a sonication technique for DNA extraction.
Iakoubova
O K
OK
Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City.
Wilke
W W
WW
Burgart
L J
LJ
Slagel
D D
DD
Wittchow
R J
RJ
Lager
D J
DJ
Heller
M J
MJ
Robinson
R A
RA
D001254:Astrocytoma / Q000235:genetics*; D001483:Base Sequence; D001932:Brain Neoplasms / Q000235:genetics*; D003550:Cystic Fibrosis / Q000235:genetics; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000235:genetics*; D005784:Gene Amplification / Q000235:genetics*; D005914:Globins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009857:Oncogenes / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016610:Tissue Embedding; D016707:Tissue Fixation
D017931:DNA Primers; D005914:Globins; D066246:ErbB Receptors