Comparison of three methods of microsatellite detection.
Examination of microsatellites is frequent in the diagnosis of cancer. Microsatellites are repeat DNA sequences scattered throughout the human genome. These repeat regions are very frequent and highly polymorphic elements. In this study we focus on dinucleotide repeats. We compared three different methods for the detection of microsatellites: use of the ABI Prism 377 fluorescence sequencer, autoradiography and silver-stained gels. DNA was extracted from various clinical samples and amplified by different polymerase chain reaction (PCR) protocols. DNA from normal and tumor tissues was analysed using each method. The fluorescence method was more sensitive than the two other methods; however, this technology is very expensive. It seems possible, when examining microsatellites on a low budget, to avoid radioactivity by using silver-stained gels as an alternative. In conclusion, we observed identical results when comparing autoradiography with the fluorescence technique. However, we observed variability in the results when interpreting a single locus comparing silver staining with autoradiography and the fluorescence technique. Classification of the tumors based on several microsatellite loci was always identical.
Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, Denmark.
M
M
Christensen
L
L
Sunde
L
L
Bolund
T F
TF
Orntoft
D001345:Autoradiography / Q000379:methods*; D003110:Colonic Neoplasms / Q000175:diagnosis / Q000235:genetics*; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000032:analysis / Q000302:isolation & purification; D004276:DNA, Satellite / Q000032:analysis / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D005819:Genetic Markers; D006801:Humans; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA / Q000379:methods*; D016622:Silver Staining / Q000379:methods*; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis / Q000235:genetics
D017931:DNA Primers; D004273:DNA, Neoplasm; D004276:DNA, Satellite; D005456:Fluorescent Dyes; D005819:Genetic Markers; D010761:Phosphorus Radioisotopes