Analysis of loss of heterozygosity in microdissected tumor cells from cervical carcinoma using fluorescent dUTP labeling of PCR products.
The use of fluorescently labeled nucleotides (F-dUTP) in the PCR for genetic typing of microsatellite polymorphisms was investigated. Microdissected tumor cells from cervical squamous cancer biopsies were compared with cells from surrounding normal tissue for loss of heterozygosity (LOH) at the mismatch repair gene hMLH1. Removal of unincorporated F-dUTP before analysis is necessary to reduce background fluorescence; ethanol-precipitation was found to be as efficient as the use of a spin column for this purpose. The gel resolution was sufficient to distinguish alleles differing by about four nucleotides. Alleles differing by only one dinucleotide repeat were possible to identify, but the ratio of alleles was difficult to assert with any reliability due to the wide peaks obtained. Single primer-pair and nested amplification systems were shown in reconstitution experiments to reliably quantitate the ratio of alleles. Of 36 cervical cancer biopsies, amplification and typing of the hMLH1 microsatellite marker was successful in 20 cases. Among 9 informative (heterozygous) biopsies, 2 (22%) were found to show LOH.
Magnusson
P K
PK
Uppsala University Biomedical Center, Sweden.
Wilander
E
E
Gyllensten
U
U
D000483:Alleles; D002872:Chromosome Deletion; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D004210:Dissection; D005260:Female; D005453:Fluorescence; D006801:Humans; D016133:Polymerase Chain Reaction; D002583:Uterine Cervical Neoplasms / Q000235:genetics*
D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate