Light-induced molecular cutting: localized reaction on a single DNA molecule.
A short focused pulse of light was used to selectively cut lambda-phage DNA molecules at specific restriction sites. Lambda DNA (48.5 kbp) was stretched and placed in a solution containing a restriction enzyme (Sma 1), caged magnesium ions (using a DM-Nitrophen complex), and a chelating agent (EDTA). When a pulse of UV light was directed at a particular location on the stretched DNA molecule, magnesium ions were released into solution. A series of binding reactions then occur in which the enzyme and the chelating agent compete for free Mg2+ ions. Since Sma 1 functions only in the presence of Mg2+, as is true of most endonucleases, the site(s) in the vicinity of the pulse (typically approximately 6 microm) were cut while other sites (three total for this DNA/enzyme pair) were not. The ratio of the concentration of the chelating agent to that of the magnesium ions was used to control the radius of this reaction zone with higher ratios leading to smaller, localized reaction areas. This optically based reaction mechanism could be useful to understand single molecule enzymatic kinetics, and when coupled with other DNA analysis techniques, this could be used to construct complex genotyping and sequencing devices that would analyze parts of single DNA molecules.
Namasivayam
Vijay
V
Department of Chemical Engineering, The University of Michigan, Ann Arbor, Michigan 48109-2136, USA.
Larson
Ronald G
RG
Burke
David T
DT
Burns
Mark A
MA
D010582:Bacteriophage lambda / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D053938:DNA Fragmentation; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D014466:Ultraviolet Rays
D004247:DNA; D004262:DNA Restriction Enzymes; D008274:Magnesium