TY - JOUR
T1 - Multiplexed DNA sequencing-by-synthesis
AU - Aksyonov, Sergei A.
AU - Bittner, Michael
AU - Bloom, Linda B.
AU - Reha-Krantz, Linda J.
AU - Gould, Ian
AU - Hayes, Mark
AU - Kiernan, Urban A.
AU - Niederkofler, Eric E.
AU - Pizziconi, Vincent
AU - Rivera, Raul S.
AU - Williams, Daniel J B
AU - Williams, Peter
N1 - Funding Information:
The advice and assistance of Tom Taylor (Arizona State University) and of Tom McCarty and Chris Gooden (Translational Genomics Research Institute) are greatly appreciated, as is the early advice on attachment chemistry issues from Michael Pirrung (North Carolina State University), Myoyong Lee (National Human Genome Research Institute [NHGRI]), and Alexander Lushnikov (Arizona State University). The authors appreciate the generous gift of cloned BRCA1 genes from Michael Erdos (NHGRI). This work was supported by the National Institutes of Health (grant R21 HG02284). Early support from the Office of the Vice-Provost for Research at Arizona State University is gratefully acknowledged. Linda Reha-Krantz acknowledges support from the Natural Sciences and Engineering Research Council (Canada) and the Alberta Heritage Foundation for Medical Research.
PY - 2006/1/1
Y1 - 2006/1/1
N2 - 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.
AB - 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.
KW - DNA
KW - Microarrays
KW - Primer extension
KW - Sequencing method
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U2 - 10.1016/j.ab.2005.10.001
DO - 10.1016/j.ab.2005.10.001
M3 - Article
C2 - 16289447
AN - SCOPUS:29144443533
SN - 0003-2697
VL - 348
SP - 127
EP - 138
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -