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Published online on July 6, 2005, 10.1073/pnas.0503809102
PNAS | July 19, 2005 | vol. 102 | no. 29 | 10012-10016
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APPLIED PHYSICAL SCIENCES
Restriction mapping in nanofluidic devices

Robert Riehn *, Manchun Lu {dagger}, Yan-Mei Wang *, Shuang Fang Lim *, Edward C. Cox {dagger}, and Robert H. Austin *, {ddagger}

Departments of*Physics and{dagger}Molecular Biology, Princeton University, Princeton, NJ 08544

Contributed by Robert H. Austin, May 12, 2005

We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100-200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg2+ and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of {approx}1.5 kbp in 1 min using single DNA molecules.

Author contributions: R.R., Y.-M.W., E.C.C., and R.H.A. designed research; R.R., M.L., and S.F.L. performed research; R.R., M.L., and E.C.C. contributed new reagents/analytic tools; R.R. and S.F.L. analyzed data; and R.R., Y.-M.W., E.C.C., and R.H.A. wrote the paper.

Freely available online through the PNAS open access option.

§ We have observed that Mg2+ concentrations of 2 mM or more lead to a quenching of fluorescence from bis-intercalating DNA dyes. We believe this is due to removal of dye from the DNA. Thus, at sufficiently high Mg2+ concentrations, intercalating dyes such as TOTO-1 or Sybr Green do not interfere with sequence-specific enzymes, because the dyes are not effectively blocking the interaction anymore.

{ddagger} To whom correspondence should be addressed. E-mail: rha{at}suiling.princeton.edu.

© 2005 by The National Academy of Sciences of the USA


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