Multiplexed DNA sequencing-by-synthesis

Sergei A. Aksyonov, Michael Bittner, Linda B. Bloom, Linda J. Reha-Krantz, Ian Gould, Mark Hayes, Urban A. Kiernan, Eric E. Niederkofler, Vincent Pizziconi, Raul S. Rivera, Daniel J B Williams, Peter Williams

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

We report a new DNA sequencing-by-synthesis method in which the sequences of DNA templates, hybridized to a surface-immobilized array of DNA primers, are determined by sensing the number of nucleotides by which the primers in each array spot are extended in sequential DNA polymerase-catalyzed nucleotide incorporation reactions, each with a single fluorescein-labeled deoxyribonucleoside triphosphate (dNTP) species. The fluorescein label is destroyed after each readout by a photostimulated reaction with diphenyliodonium chloride. A DNA polymerase with enhanced ability to incorporate, and to extend beyond, modified nucleotides is used. Self-quenching of adjacent fluorescein labels, which impedes readout of homopolymeric runs, is avoided by diluting the labeled dNTP with unlabeled reagent. Misincorporation effects have been quantified and are small; however, low-level contamination of dNTPs with other nucleotides mimics misincorporation and can produce significant false-positive signals. These impurities are removed by polymerase-catalyzed incorporation into complementary "cleaning duplexes." Here, we demonstrate the accurate sequence readout for a small array of known DNA templates, the ability to quantify homopolymeric runs, and a short sequencing example of sections of the wild-type and mutant BRCA1 gene. For a 20,000-spot array, readout rates in excess of 6000 bases per minute are projected.

Original languageEnglish (US)
Pages (from-to)127-138
Number of pages12
JournalAnalytical Biochemistry
Volume348
Issue number1
DOIs
StatePublished - Jan 1 2006

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Keywords

  • DNA
  • Microarrays
  • Primer extension
  • Sequencing method

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Aksyonov, S. A., Bittner, M., Bloom, L. B., Reha-Krantz, L. J., Gould, I., Hayes, M., Kiernan, U. A., Niederkofler, E. E., Pizziconi, V., Rivera, R. S., Williams, D. J. B., & Williams, P. (2006). Multiplexed DNA sequencing-by-synthesis. Analytical Biochemistry, 348(1), 127-138. https://doi.org/10.1016/j.ab.2005.10.001