An integrative approach for the optical sequencing of single DNA molecules.
A new approach for optically sequencing ensembles of single DNA molecules using DNA polymerase to mediate the consecutive incorporation of fluorochrome-labeled nucleotides into an array of large single DNA molecules is presented. The approach utilizes cycles of labeled fluorochrome addition, detection to count incorporations, and bleaching to reset the counter. These additions are imaged and analyzed to estimate the number of labeled additions and to correlate them on a per-locus basis along DNA backbones. Initial studies used precisely labeled polymerase chain reaction products to aid the development and validation of simple models of fluorochrome point spread functions within the imaging system. In complementary studies, nucleotides labeled with the fluorochrome R110 were incorporated into surface-elongated lambda DNA, and fluorescent signals corresponding to the addition of R110-dUTP were counted and assigned precise loci along DNA backbones. The labeled DNAs were then subjected to photobleaching and to a second cycle of addition of R110-labeled nucleotides-a second round of additions was correlated with the first to establish strings of addition histories among the ensemble of largely double-stranded templates. These results confirm the basic operational validity of this approach and point the way to the development of a practical system for optical sequencing.
Ramanathan
Arvind
A
Laboratory for Molecular and Computational Genomics, Department of Chemistry, Laboratory of Genetics, University of Wisconsin-Madison, UW-Biotechnology Center, 425 Henry Mall, Madison, WI 53706, USA.
Huff
Edward J
EJ
Lamers
Casey C
CC
Potamousis
Konstantinos D
KD
Forrest
Daniel K
DK
Schwartz
David C
DC
D004259:DNA-Directed DNA Polymerase; D003856:Deoxyuracil Nucleotides; D004867:Equipment Design; D005456:Fluorescent Dyes; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D012235:Rhodamines; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
C473629:6-carboxyrhodamine-ddUTP; D003856:Deoxyuracil Nucleotides; D005456:Fluorescent Dyes; D012235:Rhodamines; D004259:DNA-Directed DNA Polymerase