A new method for straightening DNA molecules for optical restriction mapping.
We have developed an improved method of straightening DNA molecules for use in optical restriction mapping. The DNA was straightened on 3-aminopropyltriethoxysilane-coated glass slides using surface tension generated by a moving meniscus. In our method the meniscus motion was controlled mechanically, which provides advantages of speed and uniformity of the straightened molecules. Variation in the affinity of the silanized surfaces for DNA was compensated by precoating the slide with single-stranded non-target blocking DNA. A small amount of MgCl2 added to the DNA suspension increased the DNA-surface affinity and was necessary for efficient restriction enzyme digestion of the straightened surface-bound DNA. By adjusting the amounts of blocking DNA and MgCl2, we prepared slides that contained many straight parallel DNA molecules. Straightened lambda phage DNA (48 kb) bound to a slide surface was digested by EcoRI restriction endonuclease, and the resulting restriction fragments were imaged by fluorescence microscopy using a CCD camera. The observed fragment lengths showed excellent agreement with their predicted lengths.
Yokota
H
H
Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195, USA.
Johnson
F
F
Lu
H
H
Robinson
R M
RM
Belu
A M
AM
Garrison
M D
MD
Ratner
B D
BD
Trask
B J
BJ
Miller
D L
DL
D010582:Bacteriophage lambda / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry*; D015636:Magnesium Chloride / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D015183:Restriction Mapping / Q000379:methods*; D013500:Surface Tension
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D015636:Magnesium Chloride; D004262:DNA Restriction Enzymes