Progress towards single-molecule DNA sequencing: a one color demonstration.
Single molecules of fluorescently labeled nucleotides were detected during the cleavage of individual DNA fragments by a processive exonuclease. In these experiments, multiple (10-100) strands of DNA with tetramethyl rhodamine labeled dUMP (TMR-dUMP) incorporated into the sequence were anchored in flow upstream of the detection region of an ultra sensitive flow cytometer. A dilute solution of Exonuclease I passed over the microspheres. When an exonuclease attached to a strand, processive digestion of that strand began. The liberated, labeled bases flowed through the detection region and were detected at high efficiency at the single-molecule level by laser-induced fluorescence. The digestion of a single strand of DNA by a single exonuclease was discernable in these experiments. This result demonstrates the feasibility of single-molecule DNA sequencing. In addition, these experiments point to a new and practical means of arriving at a consensus sequence by individually reading out identical sequences on multiple fragments.
Werner
James H
JH
Bioscience Division, Los Alamos National Laboratory, Mail Stop J586, Los Alamos, NM 87545, USA. jwerner@lanl.gov
Cai
Hong
H
Jett
James H
JH
Reha-Krantz
Linda
L
Keller
Richard A
RA
Goodwin
Peter M
PM
D003116:Color; D016384:Consensus Sequence; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D003911:Dextrans; D005090:Exodeoxyribonucleases / Q000737:chemistry*; D005240:Feasibility Studies; D005434:Flow Cytometry / Q000379:methods*; D011119:Polynucleotides / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012235:Rhodamines; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D003911:Dextrans; C067661:Fluoro-Ruby; D011119:Polynucleotides; D012235:Rhodamines; D004247:DNA; D005090:Exodeoxyribonucleases