High-speed and high-accuracy DNA sequencing by capillary gel electrophoresis in a simple, low cost instrument Two-color peak-height encoded sequencing at 40°C

Abstract

A low-cost DNA sequencer was constructed based on a single helium-neon laser. The two-color peak-height encoded sequencing protocol, based on the use of T7 DNA polymerase in a manganese buffer, was used to generate samples. Two termination reactions were performed. In the first, a TAMRA (applied Biosystems)-labeled primer was extended in the presence of ddATP and ddCTP. The amounts of dideoxynucleotides were adjusted to produce a 3:1 peak height ratio. Similarly, a ROX (Applied Biosystems)-labeled primer was extended in the presence of ddGTP and ddTTP; the amounts of dideoxynucleotides was adjusted to produce a 3:1 peak height ratio. The pooled fragments were separated on a 4% T LongRanger gel operated at 39°C. Over 500 bases of sequence were generated in 50 min.