AUTOMATED CYCLE SEQUENCING WITH TAQUENASE(TM) - PROTOCOLS FOR INTERNAL LABELING, DYE PRIMER AND DOUBLEX SIMULTANEOUS SEQUENCING

This paper describes automated cycle sequencing protocols for internal labeling, dye primer and ''doubles'' simultaneous sequencing using Ta quenase(TM), a new genetically modified DNA polymerase with increased thermostability. Sequencing performance both with labeled and unlabele d primer yields uniform unambiguous signals up to the resolution limit of the sequencing gels. Primer walking with internal labeling was suc cessfully performed on P1-derived artificial chromosome (PAC) construc ts with 130-kb inserts. Taquenase, a commercially available modified t hermostable sequencing enzyme (Delta 280, F667Y Taq DNA polymerase), i ncorporates a variety of fluorescent dNTPs carrying fluorescein isothi ocyanate, TexasRed(R) or Cy5(TM) labels during the cycle-sequencing pr ocess with higher efficiency than other thermostable DNA polymerases. Comparison to other modified Taq DNA polymerases suggests that the par ticular N-terminal deletion of Taquenase rather than the presence of t he F667Y mutation is responsible for the efficient incorporation and e xtension of labeled dNTPs. Taquenase makes feasible highly accurate '' doubles'' simultaneous cycle sequencing on both strands of template DN A with two internal labels or two dye-labeled primers in combination w ith the EMBL-2-dye DNA sequencing system, ARAKIS, or with two commerci al DNA sequencers. It allows up toe 2000 bases at >99% accuracy to be determined in a single reaction.