Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects.
We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 microm. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed approximately 50 microm in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43+/-0.19 ns (Cy5-dCMP), and 2.35+/-0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.
Physikalisch-Chemisches Institut, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany. sauer@urz.uni-heidelburg.de
M
M
Sauer
B
B
Angerer
W
W
Ankenbauer
Z
Z
Földes-Papp
F
F
Göbel
K T
KT
Han
R
R
Rigler
A
A
Schulz
J
J
Wolfrum
C
C
Zander
D001483:Base Sequence; D002623:Chemistry Techniques, Analytical / Q000295:instrumentation / Q000379:methods; D004247:DNA / Q000138:chemical synthesis / Q000302:isolation & purification; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D005544:Forecasting; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*
D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I