Simultaneous assessment of loss of heterozygosity at multiple microsatellite loci using semi-automated fluorescence-based detection: subregional mapping of chromosome 4 in cervical carcinoma.
Detection of loss of heterozygosity (LOH) by comparison of normal and tumor genotypes using PCR-based microsatellite loci provides considerable advantages over traditional Southern blotting-based approaches. However, current methodologies are limited by several factors, including the numbers of loci that can be evaluated for LOH in a single experiment, the discrimination of true alleles versus "stutter bands," and the use of radionucleotides in detecting PCR products. Here we describe methods for high throughput simultaneous assessment of LOH at multiple loci in human tumors; these methods rely on the detection of amplified microsatellite loci by fluorescence-based DNA sequencing technology. Data generated by this approach are processed by several computer software programs that enable the automated linear quantitation and calculation of allelic ratios, allowing rapid ascertainment of LOH. As a test of this approach, genotypes at a series of loci on chromosome 4 were determined for 58 carcinomas of the uterine cervix. The results underscore the efficacy, sensitivity, and remarkable reproducibility of this approach to LOH detection and provide subchromosomal localization of two regions of chromosome 4 commonly altered in cervical tumors.
Hampton
G M
GM
Ludwig Institute for Cancer Research, Department of Pathology, University of California at San Diego 92093-0660, USA.
Larson
A A
AA
Baergen
R N
RN
Sommers
R L
RL
Kern
S
S
Cavenee
W K
WK
D002872:Chromosome Deletion; D002874:Chromosome Mapping; D002894:Chromosomes, Human, Pair 4; D004276:DNA, Satellite / Q000235:genetics*; D005260:Female; D005453:Fluorescence; D005819:Genetic Markers; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D018895:Microsatellite Repeats / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000473:pathology
D004276:DNA, Satellite; D005819:Genetic Markers