Sequencing Reactions for the Applied Biosystems 373AAutomated DNA Sequencer

Efficient completion of large DNA sequencing projects has been greatly facilitated by the development of fluorescence-based dideoxynucleotide sequencing chemistries and instruments for real-time detection of fluorescence-labeled DNA fragments during gel electrophoresis (1 –6 ;see Chapter 1 ). Besides eliminating the use of radioisotopes, these systems automate the task of reading sequences and provide computerreadable data that may be directly analyzed or entered into a sequence assembly engine. In this chapter, we describe DNA sequencing using the first commercially-available fluorescent instrument, the Applied Biosystems, Inc. Model 373A Automated DNA Sequencer. This sequencer, originally introduced in 1987 as the Model 370A, utilizes a multi-spectral approach in which four distinct fluorescent tags are detected and differentiated in a single lane on the sequencing gel (6 ). The four tags are incorporated during the DNA sequencing reactions and may be present on either the 5′ end of the sequencing primer (”dye-primers”) or on the dideoxynucleotide triphosphate (”dye-terminators”). Since the 373A requires only one lane per sample, up to thirty-six samples may be analyzed per run. By comparison, the DuPont Genesis system currently supports only a four-dye, one-lane dye-terminator chemistry and allows analysis of up to twelve samples per run. The Pharmacia ALF sequencer supports only a one-dye, fourlane approach with dye-labeled primer, and can analyze up to ten samples per run. The ability of the 373A to use both dye-primer and dyeterminator chemistries greatly facilitates the completion of projects that require both shotgun and primer-directed sequencing phases