Two-label peak-height encoded DNA sequencing by capillary gel electrophoresis: three examples

摘要: We report a modification to the peak-height encoded DNA sequencing technique of Tabor and Richardson. As in original protocol, reaction uses modified T7 polymerase with manganese rather than magnesium produce very uniform incorporation each dideoxynucleoside. To improve accuracy, two fluorescently labeled primers are employed separate reactions. an example, one FAM-labeled primer dideoxyadenosine triphosphate dideoxycytosine triphosphate; concentrations ddATP ddCTP adjusted 2:1 variation relative intensity fragments. The second TAMRA dideoxythymidine dideoxyguanidine ddTTP ddGTP pooled products separated by capillary gel electrophoresis identified three different detector systems. Use peak height ratio typically produces accuracy 97.5% for first 350 bases; 3:1 improves 99.5% 400 bases. For these experiments, is performed at electric field 200 V/cm; hours required fragments up nucleotides length.