Multiplexed DNA sequencing-by-synthesis

• Published in: Analytical biochemistry 2006, Vol.348 (1), p.127-138
• Main Authors: Aksyonov, Sergei A., Bittner, Michael, Bloom, Linda B., Reha-Krantz, Linda J., Gould, Ian R., Hayes, Mark A., Kiernan, Urban A., Niederkofler, Eric E., Pizziconi, Vincent, Rivera, Raul S., Williams, Daniel J.B., Williams, Peter
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; DNA; DNA-Directed DNA Polymerase; Genes, BRCA1; Microarrays; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis - methods; Polymerase Chain Reaction; Primer extension; Sensitivity and Specificity; Sequence Analysis, DNA - methods; Sequencing method
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2005.10.001

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.