Rapid detection of allele loss in colorectal tumours using microsatellites and fluorescent DNA technology.
In order to investigate allele loss in colorectal tumours we have developed a rapid technique which overcomes most of the problems associated with radioactive Restriction Fragment Length Polymorphism (RFLP) analysis of allele loss. We utilise microsatellite length polymorphisms which are highly informative and are closely linked to loci of interest. Sequences containing microsatellites can be amplified from normal and tumour DNA pairs by a polymerase chain reaction (PCR) in which one of the primers is fluorescently labelled. This enables us to detect the products on polyacrylamide gels run on an automated DNA sequencer using dedicated software, by which results are automatically quantitated in terms of peak size, height, and area. Using this technique we have analysed 26 normal tissue: cancer pairs for allele loss at two loci linked to the adenomatous polyposis coli (APC) gene on chromosome 5q. Repeated assays yielded identical results for each pair. Allele loss was found in 10 out of 25 informative samples (40%).
Cawkwell
L
L
Academic Unit of Pathological Sciences, University of Leeds, UK.
Bell
S M
SM
Lewis
F A
FA
Dixon
M F
MF
Taylor
G R
GR
Quirke
P
P
D000230:Adenocarcinoma / Q000235:genetics*; D000483:Alleles; D002872:Chromosome Deletion; D002895:Chromosomes, Human, Pair 5 / Q000502:physiology; D015179:Colorectal Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005453:Fluorescence; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
D004273:DNA, Neoplasm