A quantitative study of optical mapping surfaces by atomic force microscopy and restriction endonuclease digestion assays.
Many new techniques in biomolecular chemistry and genomic analysis require the immobilization of molecular reagents on specially prepared surfaces. However, the process of molecular fixation often interferes with or precludes the use of standard in vitro biochemical assays. Optical mapping is an emergent technology for genomic analysis which relies on the biochemical activity of DNA fixed to silanized glass surfaces. Optical mapping surfaces have been shown to be compatible with restriction endonucleases and a variety of DNA polymerases. The essential properties of biochemically active surfaces are poorly understood in most of the current technologies which utilize molecular fixation, including optical mapping. The purpose of this study is to use the powerful technique of atomic force microscopy, in combination with informative enzymatic assays, to correlate biochemical activity with microscopic surface structure. The results presented provide meaningful insight into the effect of surface preparation on the biochemical accessibility of surface-bound molecules. Novel analysis which may facilitate the automation of optical mapping is presented.
Reed
J
J
Department of Chemistry, W. M. Keck Laboratory for Biomolecular Imaging, New York University, New York 10003, USA.
Singer
E
E
Kresbach
G
G
Schwartz
D C
DC
D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D005821:Genetic Techniques; D006868:Hydrolysis; D007202:Indicators and Reagents; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods*; D015183:Restriction Mapping / Q000295:instrumentation / Q000379:methods
D007202:Indicators and Reagents; D004247:DNA; D004262:DNA Restriction Enzymes