Light-Induced Molecular Cutting:  Localized Reaction on a Single DNA Molecule

Abstract

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.

Exploring both sequence detection and restriction endonuclease cleavage kinetics by recognition site via single-molecule microfluidic trapping

“…S1 in reference 21). In addition, we do not need to immobilize the DNA on a surface, so we eliminate the potential steric and diffusion limitations mentioned above; 30, 31, 43 furthermore, in our device the DNA or the fragments following the first cleavage event may be easily recovered for downstream analysis or processing.…”

Polymer-monovalent salt-induced DNA compaction studied via single-molecule microfluidic trapping

“…35 Device masters were fabricated to a 130-μm depth using SU-8 2100 resist (MicroChem). PDMS (Sylgard 184, Dow Corning) molds were cast from the master, and inlets and outlets were created using a 16-gauge blunt-tipped needle.…”

Single‐Molecule Studies on DNA and RNA

“…caged calcium and with a sufficiently sharply focused laser, it is even possible to select the specific part of a long DNA molecule from which the reaction should proceed. [46] For practical single-molecule DNA typing it is often not necessary to stretch the molecules. Restriction endonucleases also work on collapsed DNA molecules (see Figure 5).…”