AmpliTaq DNAPolymerase, FS is the sequencing enzyme used in ABI PRISM cycle sequencing kits. It is a mutant form of Thermus aquaticus (Taq) DNA polymerase and contains a point mutation in the active site, replacing phenylalanine with tyrosine at residue 667 (F667Y). This mutation results in less discrimination against dideoxynucleotides, and leads to a much more even peak intensity pattern [172].
AmpliTaq DNA Polymerase, FS also contains a point mutation in the amino terminal domain, replacing glycine with aspartate at residue 46 (G46D), which removes almost all of the 5’→3’ nuclease activity. This eliminates artifacts that arise from the exonuclease activity. The enzyme has been formulated with a thermally stable inorganic pyrophosphatase that cleaves the inorganic pyrophosphate (PPi) byproduct of the extension reaction and prevents its accumulation in the sequencing reaction.
In the presence of high concentrations of PPi the polymerisation reaction can be reversed [173], a reaction called pyrophosphorolysis. In this reaction, a nucleoside monophosphate is removed from the extension product with the addition of PPi to form the nucleoside triphosphate. In a sequencing reaction, if a dideoxynucleotide is frequently removed at a particular position and replaced by a deoxynucleotide, eventually there is little or no chain termination at that location. This results in a weak or missing peak in the sequence data [174].
With dye primer labelling, primers are tagged with four different fluorescent dyes. Labelled products are generated in four separate base-specific reactions. The products from these four reactions are then combined and loaded into a single gel lane. Features of dye-labelled primer reactions:
With dye terminator labelling, each of the 4 dideoxy terminators (ddNTPs) is tagged with a different fluorescent dye. The growing chain is simultaneously terminated and labelled with the dye that corresponds to that base. Features of dye-labelled terminator reactions:
© 2001 Alexander Binder