Five useful approaches for generating more valid gel images of loss of heterozygosity and clonality analysis with an automated 377 DNA sequencer.
The recently introduced fluorescence-based gene scan system for assessment of loss of heterozygosity (LOH) and clonality with an automated DNA sequencer has several advantages over the traditional method. However, the production of gel images with this system is subjected to more technical challenges, including the interference of autofluorescence, weaker and less consistent signals that result from the restricted well size and difficulties in sample loading. To minimize the impact of these technical difficulties, several unique strategies were used, including the following: elimination of fabrics or paper towels in the cleaning of gel plates and containers; use of a modified loading buffer; use of more concentrated gels; use of an innovative apparatus to clean gel wells before and after the prerun; and covering the black printer cartridge with a sheet of scotch tape. With these strategies, the authors have been able to consistently obtain gel images that can be presented as either densitometric graphs or as band patterns for direct visual assessment.
Man
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Department of Gynecologic and Breast Pathology, The Armed Forces Institute of Pathology, Washington, DC 20306-6000, USA.
Moinfar
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Bratthauer
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Tavassoli
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D001322:Autoanalysis / Q000379:methods*; D002999:Clone Cells; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019656:Loss of Heterozygosity; D016133:Polymerase Chain Reaction