The dideoxy method for DNA sequencing, developed in the Seventies by Sanger, takes advantage of the ability of DNA polymerase to incorporate analogues of nucleotide bases by using 2´,3´-dideoxynucleotides (ddNTPs) as substrates. When a dideoxynucleotide is incorporated at the 3´ end of the growing chain, chain elongation is terminated selectively at A, C, G, or T because the ddNTP lacks a 3´-hydroxyl group. Since its discovery, the method has undergone many improvements regarding labeling technology, chemistry and instrumentation, nevertheless, the base protocol remains essentially unchanged.
The BigDye® Terminator kit from Applied Biosystems uses four different fluorescent dyes to label ddNTPs, which are added sequentially to the primer through a cycle sequencing reaction. The kit provides all required reagents for the sequencing procedure in a reaction-ready, pre-mixed format. These reagents are suitable for sequencing of single-stranded or double-stranded DNA templates, PCR products, and large templates. Buffer composition is designed to allow a single tube reaction for each primer, producing a series of molecules of different length, each one terminating and labeled at a different base. The ratio of dNTPs/ddNTPs is calculated so that termination is obtained at least once for every position of the template. Reaction products can then be run in an automated sequencer to obtain the final sequence.
I mainly use cycle sequencing to analyze viral and human genes for mutation and polymorphism identification. My templates chiefly consist of PCR products obtained through standard or nested-PCR amplification. I chose a dye-terminator chemistry because it allows a single-tube reaction by using four different dyes that can be detected simultaneously. Moreover, the AmpliTaq DNA Polymerase FS used in this kit assures a very high degree of fidelity, which is a good feature when dealing with mutation detection. BigDye® dyes contain a fluorescein donor dye linked to one of four different dichlororhodamine acceptor dyes. Because the linker affords extremely efficient energy transfer between the two dyes, BigDye® terminators are 2-3 times brighter and have narrower emission spectra than previous chemistries, resulting in brighter signals and decreased noise. As a result, the sequencing mixes are able to give longer, more accurate read lengths and small volume reactions can be performed. In my experiments, I optimized the protocol proposed by Applied Biosystems, reducing all concentrations and working on smaller volumes in order to perform more reactions without loss in quality of results. Even at low titers, sequence data obtained with Applied Biosystems BigDye® Terminator v3.1 kit are still good and neat, proving the high quality of the system and its reliability.
Overall, this kit is an excellent product. It is very easy to use and highly efficient. Even when used at very low concentrations and thawed repeatedly, it still gives good and reproducible results. I feel so confident about Applied Biosystems BigDye® that for routine procedures I seldom use a positive control, since dubious reactions are very rare.
Dr. Andrea Galli
University of Milan
Dept of Clinical Sciences