Sanger sequencing, the process used for automated sequencing, requires a DNA template to be amplified by the Polymerase Chain Reaction (PCR). Despite similarities between the processes, a sequencing amplification is different than basic PCR.
Sanger sequencing utilizes linear amplification
PCR produces millions of copies of a DNA region from a single copy of template DNA. Each copy produced during PCR in one cycle becomes a new template for the next cycle. PCR uses forward and reverse primers. The forward primer anneals to a complimentary site on one strand of DNA and extends toward the reverse primer. In turn, the reverse primer similarly extends towards the forward primer. What results is a copy of the desired region of DNA to be amplified. The new copy contains priming sites so it can be used as a template for future amplifications (figure 1). One copy of the original template produces two copies; two copies produce four in the next cycle; and so on. A twenty-five cycle PCR will produce 2E24 copies from a single template.
Sanger sequencing uses one primer instead of two. The amplification process copies one strand but not the reverse strand. The copy is the same direction as the primer and cannot be used as a template for later cycles. All amplification is directly from the original template DNA in the reaction. Therefore, amplification is linear, not exponential. It is the reason that Sanger sequencing amplification must include sufficient copies of the original template DNA to be visualized by automated sequencing equipment (figure 2).
Dideoxynucleotide bases are included in Sanger sequencing
The components of basic PCR include buffer, the enzyme Taq polymerase, deoxynucleotides (dNTPs), template, and forward and reverse primers. Sanger sequencing includes an additional component called dideoxynulceotides (ddNTPs). The ddNTPs are terminating bases that include a fluorescent tag for automated sequencing equipment. For this reason, Sanger sequencing is also called dye-terminator sequencing. During amplification, the ddNTPs will randomly sit on the DNA template and terminate the extension. The dNTPs sit on the remaining templates and continue extending. The end product is a size ladder of PCR products that increase by a single base (figure 2). Each terminating base is tagged with fluorescent dye. This dye provides a unique color representing the A, G, C, and T bases in DNA.
The DNA ladder is separated by electrophoresis
Once PCR is complete, the products produced in the Sanger reaction are loaded on an automated slab gel or capillary analyzer. Products will separate by size with smaller products moving faster through the medium. As the products near the end of the medium, the fluorescent tags are excited by light and recorded to a computer with a digital camera (figure 3). The computer records the color for each band and assigns the correct base to complete dye-terminator sequencing.