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 degrees C.
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 degrees C. Over 500 bases of sequence were generated in 50 min.
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Department of Chemistry, University of Alberta, Edmonton, Canada.
Arriaga
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Chen
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Dovichi
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NJ
D002197:Capillary Action; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation* / Q000379:methods* / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature
D017931:DNA Primers; D005456:Fluorescent Dyes