Chemiluminescent DNA sequencing with multiplex labeling

Abstract

Chemiluminescent detection techniques provide a sensitive, nonradioactive method for DNA sequencing. Standard Sanger dideoxy DNA sequencing reactions are initiated with biotinylated primers, separated by gel electrophoresis, transferred to nylon membrane and detected utilizing chemiluminescent 1,2-dioxetane substrates for alkaline phosphatase. A multiplex-labeling method was developed to permit detection of several overlapping sets of DNA sequence information on a single membrane, thereby increasing the productivity of a single gel electrophoretic separation. Primers labeled with different haptens at the 5' end were used to perform separate sequencing reactions. These were mixed together prior to electrophoresis, and the individual sequencing products sequentially detected using hapten-specific reagents. We incorporated primers labeled with biotin, digoxigenin, 2,4-dinitrophenyl or fluorescein, each consecutively detected with a hapten-specific alkaline phosphatase conjugate and CSPD 1,2-dioxetane chemiluminescent substrate. To further increase the amount of DNA sequence data that can be obtained from a single membrane, a direct transfer electrophoresis apparatus was used for simultaneous separation of the DNA sequencing reactions and membrane transfer. The resulting increased separation of the high molecular weight fragments yields 350-450 bp of readable DNA sequence data from each template. Chemiluminescent detection of overlapping sets of DNA sequencing reactions utilizing multiplex labeling, combined with direct transfer electrophoresis, provides an efficient, nonradioactive method for DNA sequencing.