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

Hui Lu, Edgar Arriaga, Yong Chen Da, Norman J. Dovichi

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)497-501
Number of pages5
JournalJournal of Chromatography A
Volume680
Issue number2
DOIs
StatePublished - Oct 7 1994

Bibliographical note

Funding Information:
This work was supported in part by the Department of Energy (DOE)-Human Genome Initiative (USA) grant number DE-FGOZ 91ER61123. Support by DOE does not constitute an endorsemento f the views expressedi n this article. This work was also supportedb y the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Department of Chemistry of the University of Alberta. N.J.D. acknowledgesa Steacie fellowship from NSERC.

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