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

Abstract

We have evaluated the use of near infrared (NIR) dye-labeled AcycloTerminators™ for sequencing large-insert clones such as BACs. These fluorescent acyclic analogs (AcyNTPs) were labeled with a polymethine carbocyanine dye (IRD800) through a propargylamino linker to the purine or pyrimidine base. Thermo Sequenase^® DNA polymerase incorporated these non-nucleotide terminators with the same efficacy and fidelity as traditional dideoxynucleotides.

Previous studies have suggested that only terminators labeled with energy-transfer dyes provide sufficient sensitivity for the direct sequencing of high molecular weight BAC DNA since template molar equivalents are limited. However, AcyNTPs tagged with a single NIR cyanine dye were used in cycle sequencing reactions with 2-3 µg of high quality large-template DNA, increased amounts of primer (up to 10 pmole), and additional thermal cycles (>60 cycles) to produce long, accurate sequence reads (Q value >20 for 800-1100 bases). Direct sequencing of BAC clones with custom primers will facilitate gap closure in large-scale projects, especially for difficult regions that are resistant to cloning and various PCR methods.