(1) Cycle sequencing
The term "cycle sequencing" is odd nomenclature for this step. Although it involves thermal cycling, just like PCR, "sequencing" technically occurs in the second and third steps. Unlike PCR, only one primer is used (i.e. the sequencing primer), so the reaction is linear rather than exponential. After each round of denaturation and annealing, template is copied via primer extension. dNTPs are in excess, but the reaction mix also includes fluorescently-labeled dideoxynucleotides (ddNTPs). ddTTP, ddCTP, ddATP, and ddGTP each have a unique fluorescent marker and cause early chain termination. Thus, the reaction produces end-labeled, variable-length extension products.
This step is the most labor-intensive, as individual samples are manually transferred to wells of a 96-well PCR plate according to a predetermined plate map (see below). The map is organized by order # and sample # (e.g. 140037.1, 140037.2, etc; highlighted yellow). This is why we GREATLY appreciate customers labeling their samples with ORDER # and SAMPLE # only! BigDye is then added to the wells, in addition to primer and/or DTB (if requested). It takes us about 30-45 minutes to setup a full 96-well plate of individual samples.
(2) Capillary electrophoresis
(3) Data analysis