Below is a brief explanation of each step at a level we recommend understanding. at right.(1) Cycle sequencingThe 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.
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(2) Capillary electrophoresis
This step is analogous to agarose gel electrophoresis of DNA. However, the DNA migrates through a micro-capillary containing an expensive liquid polymer capable of separating DNA fragments at a resolution of 1 base pair. We have two DNA Analyzers, one with 48 capillaries (Cinderella) and one with 96 capillaries (Snow White). An example of a capillary array is pictured at right.(3) Data analysis
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