AmpliTaq ® DNA Polymerase, FS Dye-Terminator Sequencing: Analysis of Peak Height Patterns

Taq DNA polymerases in which the phenylalanine is substituted by a tyrosine at position 667 (Taq F667Y) are members of a new class of DNA polymerases that incorporate chain-terminating dideoxyribonucleoside triphosphates (ddNTPs) much more efficiently than the wild-type Taq DNA polymerase. Improved incorporation of ddNTPs into DNA during cycle sequencing using AmpliTaq DNA polymerase, FS (Taq-FS, a member of the Taq F667Y family), and dye-labeled primers results in nearly uniform peak heights in the sequencing trace. This is not the case when dye-labeled ddNTPs are used in Taq-FS cycle sequencing reactions. While the rate of dye-terminator incorporation is more efficient with Taq-FS, the peak pattern is still highly variable and different from that produced by the wild-type enzyme. We have systematically examined pairs of sequence-tagged sites that vary at only a single nucleotide to determine how base changes influence the peak heights of neighboring bases in sequencing traces generated by the Taq-FS dye-terminator chemistry. In 31 of 64 possible 3-base windows (48%), we find that the peak height of a particular base can be predicted by knowing just one or two bases 5' to the base in question. We have also compared and contrasted the peak patterns produced by the Taq-FS enzyme with those previously identified for the wild-type enzyme. Establishing the patterns in peak heights within local sequence contexts can improve the accuracy of base-calling and the identification of polymorphisms/mutations when using the Taq-FS dye-terminator cycle-sequencing chemistry.