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

Introduction

Sequencing very long (i.e. >50 Kb) dsDNA molecules such as Bacterial Artificial Chromosome (BAC) clones can be problematic. The molecular length of these types of samples introduces difficulties with sample handling due to high viscosity and also causes problems with primer binding and extension. Previous studies have suggested that only energy-transfer dye terminators can provide sufficient sensitivity for direct sequencing of high molecular weight BAC DNA due to the limited number of molar equivalents of template DNA that can be included in a reaction. However, with only a few modifications to the standard LI-COR sequencing protocols, we have been able to successfully and repeatedly achieve long, high-quality read lengths on BACs. The combination of smaller amounts of DNA template with IR-labeled AcycloTerminator™ mixtures and modified cycling procedures resulted in long accurate sequencing results on BACs.