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 Citations (Scopus)

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

Fingerprint

DNA Sequence Analysis
Nucleotides
Fluorescein
Deoxyribonucleosides
DNA
DNA-Directed DNA Polymerase
Labels
Immobilized Nucleic Acids
BRCA1 Gene
DNA Primers
Oligonucleotide Array Sequence Analysis
Quenching
Cleaning
Contamination
Genes
Impurities
triphosphoric acid

Keywords

  • DNA
  • Microarrays
  • Primer extension
  • Sequencing method

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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

In: Analytical Biochemistry, Vol. 348, No. 1, 01.01.2006, p. 127-138.

Research output: Contribution to journalArticle

Aksyonov, SA, Bittner, M, Bloom, LB, Reha-Krantz, LJ, Gould, I, Hayes, M, Kiernan, UA, Niederkofler, EE, Pizziconi, V, Rivera, RS, Williams, DJB & Williams, P 2006, 'Multiplexed DNA sequencing-by-synthesis', Analytical Biochemistry, vol. 348, no. 1, pp. 127-138. https://doi.org/10.1016/j.ab.2005.10.001
Aksyonov SA, Bittner M, Bloom LB, Reha-Krantz LJ, Gould I, Hayes M et al. Multiplexed DNA sequencing-by-synthesis. Analytical Biochemistry. 2006 Jan 1;348(1):127-138. https://doi.org/10.1016/j.ab.2005.10.001
Aksyonov, Sergei A. ; Bittner, Michael ; Bloom, Linda B. ; Reha-Krantz, Linda J. ; Gould, Ian ; Hayes, Mark ; Kiernan, Urban A. ; Niederkofler, Eric E. ; Pizziconi, Vincent ; Rivera, Raul S. ; Williams, Daniel J B ; Williams, Peter. / Multiplexed DNA sequencing-by-synthesis. In: Analytical Biochemistry. 2006 ; Vol. 348, No. 1. pp. 127-138.
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