Sequencing BAC DNA With Near-Infrared
Fluorescent Non-Nucleotide Terminators

Stephen C. Roemer, Vince R. Boveia, Craig M. Johnson and D. Michael Olive
LI-COR Inc., Biotechnology, 4647 Superior Street, Lincoln, NE 68504

Conclusions

AcyNTPs showed performance equivalent to IR-labeled ddNTP and consistently produced read lengths over 1000 bases with a high degree of accuracy. We have further demonstrated the utility of IR-labeled acyNTPs for producing long, high-quality reads on BAC clones. The data shown here for the IR-labeled acyNTPs, combined with the LI-COR 4200L, are the longest read lengths on BAC clones reported to date. Energy transfer terminators, while producing a signal intensity sufficient to allow short-to-moderate read lengths on BAC clones, have not approached the results presented here for the IRDye® infrared dye-labeled AcycloTerminators.

BAC sequencing with dye primers can sometimes be further facilitated by the use of different DNA polymerases, which may differ in their ability to read through difficult sequences. As shown here, SequiTherm EXCEL™ II produced significantly better results than Thermo Sequenase^® on three particularly troublesome BAC clones. The ability to produce long, accurate reads on BAC clones should facilitate gap closure, reducing both the time and labor costs of genome finishing projects.