Multiplexed DNA sequencing-by-synthesis.

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.

Polymerases:

Topics:

Biotech Applications, Methods

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T4 D112AE114A/L412M Aksyonov SA2006 Application name Sequencing

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