PCR amplification: 4 tubes contain the following: :dNTPs, a specific ddNTP, DNA polymerase, primer sequence. ddNTPs lack a 3'OH group (labeled with a dye) which terminates elongation of strand synthesis. dNTPs will extend the strand synthesis until it incorporates a ddNTP. Therefore multiple strands of varying length with be generated from a single tube. (2) Analysis of PCR amplicons by capillary electrophoresis. We want to examine the terminal ends of each generated strand. Separation of strands is due to fused silica capillary with a 25-100um diameter. The high surface area to volume ratio allows dissipating heat efficiently, allowing the use of a high electric field that can decrease run time and improve resolution. (3) Base-calling: A fluorescent signal is given off and is recorded at the output of the capillary tube throughout the run, which is analyzed by computer to determine the DNA sequence referred to as base calling: a nucleotide is assigned to its peak based on its fluorescent spectrum.*##**##*
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