This talk will summarize our progress on the development of a method

to sequence single DNA fragments.  Our approach is based on

single-molecule fluorescence detection [1] and consists of: labeling

the different nucleotides types with distinguishable fluorophores;

polymerase incorporation of the labeled nucleotides into a strand of

DNA; attachment of one end of the DNA fragment to a suitable support;

movement of the supported DNA into a flow stream to cause the DNA

fragment to stretch out into the flow; exonuclease digestion of the

free end of the fluorescently-labeled DNA strand to sequentially

release the fluorophore-labeled nucleotides into the flow stream and

efficient single-molecule detection and identification of the

fluorophores in the ordered sample stream to read out the

sequence. [2,3] The method has the potential for sequencing DNA

fragments tens of thousands of bases in length, in contrast to current

gel-electrophoresis-based sequencing methods with read lengths limited

to approximately 1000 bases (1 kb).  We have made considerable

progress towards a demonstration of single DNA fragment sequencing.

Up to three fluorophore-labeled nucleotide types have been

incorporated into strands of DNA 2-7 kb in length.  Only two of the

nucleotide types need to be labeled at once; DNA can be sequenced in

multiple passes using all combinations of labeling of the nucleotide

types with two distinguishable labels. [4] Exonuclease turnover rates

of ~50 and ~1 nucleotides per second have been measured at room

temperature for Esherichia coli Exonuclease I and Exonuclease III,

respectively, on fluorescently-labeled DNA.  Multiple fragments of

labeled DNA have been attached to polystyrene microspheres.

Individual, DNA-laden microspheres have been optically trapped in flow

~30 microns upstream of a picoliter single-molecule fluorescence

detection volume [5].  Exonuclease digestion of fluorescently-labeled

DNA fragments attached to optically trapped microspheres has been

monitored by the detection of individual, fluorophore-labeled

nucleotides downstream of the microsphere.  We have detected single,

fluorophore-labeled nucleotides cleaved from as few as ~100 fragments

of DNA held on an optically trapped microsphere.  We have also used this capability to measure the turnover kinetics and to confirm

the processivity of Exonuclease I on single-stranded,

fluorescently-labeled DNA.  Rhodamine-6G (R6G) and

tetramethylrhodamine (TMR) labeled nucleotides (R6G-dCMP and

TMR-dUMP), released by exonuclease digestion of labeled DNA fragments,

have been detected and identified at the single-molecule level [6] by

correlated measurements of fluorescence intensity and lifetime using a

single excitation wavelength and detection channel [7].  This work is

supported by the United States Department of Energy, Office of

Biological and Environmental Research.

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References

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[1] Keller, R.A.; Ambrose, W.P.; Goodwin, P.M.; Jett, J.H.; Martin,

J.C.; Wu, M.  ``Single-molecule fluorescence analysis in solution,''

Appl. Spectrosc., 50:12A-32A (199
6).

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[2] Goodwin, P.M.; Cai, H.; Jett, J.H.; Ishaug-Riley, S.L.; Machara,

N.P.; Semin, D.J.; Van Orden, A.; Keller, R.A. ``Application of single-molecule detection to DNA sequencing,'' Nucleos. Nucleot., 16:543-550 (199
7).

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[3] Jett, J.H.; Keller, R.A.; Martin, J.C.; Moyzis, R.K.; Ratliff,

R.L.; Shera, E.B; Stewart, C.C. ``Method for Rapid Base Sequencing in

DNA and RNA,'' US Patent #4,962,037 (199
0).

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[4] Jett, J.H.; Keller, R.A.; Martin, J.C.; Posner, R.G.; Marrone,

B.L.; Hammond, M.L.; Simpson, D.J. ``Method for Rapid Base Sequencing in DNA and RNA with Two Base Labeling,'' US Patent #5,405,747 (199
5).

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[5] Machara, N.P.; Goodwin, P.M.; Enderlein, J.; Keller, R.A. ``Efficient detection of single molecules eluting off an optically trapped microsphere,'' Bioimaging, 6:33-42

(199
8).

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[6] Werner, J.H.; Cai, H.; Goodwin, P.M.; Keller, R.A. ``Current status of DNA sequencing by single molecule detection,'' Proc. SPIE,

3602:355-366 (199
9).

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[7] Van Orden, A.; Machara, N.P.; Goodwin P.M.; Keller,

R.A. ``Single-molecule identification in flowing sample streams by

fluorescence burst size and intraburst fluorescence decay rate,''

Anal. Chem., 70:1444-1451 (199
8).