PubMed · 12904642
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
Andrew Preston. 2003. Choosing a cloning vector.. https://doi.org/10.1385/1-59259-409-3%3A19
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.
Lambda exonuclease processively degrades one strand of duplex DNA, moving 5'-to-3' in an ATP-independent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGAT TCT, was identified by gel electrophoresis. Correlating single-molecule dwell positions with sequence independently identified the motif GGCGA. This sequence is found in the left lambda cohesive end, where exonuclease inhibition may contribute to the reduced recombination efficiency at that end.
Carbon nanotubes are nanometer-scale materials with important properties, but their use in nanofabrication will require further development of methods for controlled positioning at well-defined locations on surfaces. We have devised an approach for specifically localizing single-walled carbon nanotubes (SWNTs) onto 1-pyrenemethylamine (PMA)-decorated lambda-DNA molecules aligned on Si surfaces. PMA is used as a bridging compound because its amine group is attracted electrostatically to the negatively charged phosphate backbone of DNA, while the pyrenyl group in PMA interacts with SWNT surfaces through pi-stacking forces. From a total of 60 atomic force microscopy images obtained on three different substrates, we determined that 63% of SWNTs observed on the surfaces were anchored along DNA, and these nanotubes covered approximately 5% of the total DNA length. DNA-templated nanopositioning offers intriguing possibilities for the bottom-up assembly of materials at the nanometer scale.