Multiplexed DNA sequencing-by-synthesis.
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.
Aksyonov
Sergei A
SA
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA.
Bittner
Michael
M
Bloom
Linda B
LB
Reha-Krantz
Linda J
LJ
Gould
Ian R
IR
Hayes
Mark A
MA
Kiernan
Urban A
UA
Niederkofler
Eric E
EE
Pizziconi
Vincent
V
Rivera
Raul S
RS
Williams
Daniel J B
DJ
Williams
Peter
P
D000595:Amino Acid Sequence; D004259:DNA-Directed DNA Polymerase; D019398:Genes, BRCA1; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
D004259:DNA-Directed DNA Polymerase