Assessment of allele dosage at polymorphic microsatellite loci displaying allelic imbalance in tumors by means of quantitative competitive-polymerase chain reaction.
Analysis of allelic imbalance at polymorphic marker loci is usually employed to identify chromosomal regions affected by recurrent aberrations in tumor genomes. Such regions are likely to harbor genes involved in the onset and/or progression of cancer. Although often used to identify regions of loss of heterozygosity caused by deletions/rearrangements near tumor suppressor gene loci, allelic imbalance can also reflect regional amplification, indicating the presence of oncogenes. It is difficult to tell these two situations apart after ordinary polymerase chain reaction (PCR), but here we describe a method that distinguishes allelic loss from allelic gain. The level of allelic imbalance was determined by quantitative PCR (QPCR) in the presence of an internal control DNA that displayed a third allele at the locus studied. To validate the efficiency of allele quantitation, we analyzed an amplified region in a set of rat fibrosarcomas. In four tumor samples with amplification of the Met oncogene, we could show with QPCR that there was amplification of one of the alleles at a microsatellite marker located close to Met. QPCR may be useful for cancer studies because experiments may be predesigned for using either suitable microsatellite markers or the abundant and polymorphic poly-A tails of rodent identifier sequences.
Sjöling
Asa
A
Department of Cell and Molecular Biology-Genetics, Box 462, SE-405 30 Göteborg, Sweden. asa.sjoling@microbio.gu.se
Walentinsson
Anna
A
Nordlander
Carola
C
Karlsson
Asa
A
Behboudi
Afrouz
A
Samuelson
Emma
E
Levan
Göran
G
Röhme
Dan
D
D022981:Allelic Imbalance; D000818:Animals; D005354:Fibrosarcoma / Q000235:genetics*; D018628:Gene Dosage; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D051381:Rats; D011919:Rats, Inbred Strains