How It Works
In general, automated Sanger sequencing involves three steps: (1) cycle sequencing, (2) capillary electrophoresis, and (3) data analysis. We use this important terminology throughout the Wiki. Below is a brief explanation of each step at a level we recommend understanding. The whole process is illustrated diagrammatically in the figure at right.
(1) Cycle sequencing
The term "cycle sequencing" can be confusing. It involves thermal cycling in a PCR machine, just like PCR. However, 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, the 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. Analysis of these extension products during steps (2) and (3) will ultimately reveal the sequence.
This step is the most labor-intensive, as individual samples are manually transferred to wells of a 96-well PCR plate according to a generated 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