Biomedical subjects

R J Warmack

Publications and source records attributed to R J Warmack.

10 recordsLinked to original sources

PubMed1997

Mapping individual cosmid DNAs by direct AFM imaging.

Individual cosmid clones have been restriction mapped by directly imaging, with the atomic force microscope (AFM), a mutant EcoRI endonuclease site-specifically bound to DNA. Images and data are presented that locate six restriction sites, predicted from gel electrophoresis, on a 35-kb cosmid isolated from mouse chromosome 7. Measured distances between endonuclease molecules bound to lambda DNA, when compared to known values, demonstrate the accuracy of AFM mapping to better than 1%. These results may be extended to identify other important site-specific protein-DNA interactions, such as transcription factor and mismatch repair enzyme binding, difficult to resolve by current techniques.

Animals↗

PubMed1996

Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.

Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.

Binding Sites↗

PubMed1994

Stretched DNA structures observed with atomic force microscopy.

Double-stranded DNA molecules are occasionally found that appear to be straightened and stretched in atomic force microscope (AFM) images. Usually pBS+ plasmid and lambda DNA show relaxed structures with bends and kinks along the strands and have measured contour lengths consistent to about 5-7%; they also appear not to cross over each other, except in very high concentrations. The anomalous molecules observed here, compared with the majority of molecules in the preparation, show contour lengths increased by as much as 80% and have measured heights of about half that of normal relaxed DNA. Some molecules also appear to be in transition between stretched and relaxed forms. These observations are consistent with an uncoiling of the DNA helix without breakage of the covalent bonds in the deoxyribose-phosphate backbone.

Bacteriophage lambda↗

PubMed1992

Immobilization of DNA for scanning probe microscopy.

Reproducible scanning tunneling microscope and atomic force microscope images of entire molecules of uncoated plasmid DNA chemically bound to surfaces are presented. The chemically mediated immobilization of DNA to surfaces and subsequent scanning tunneling microscope imaging of DNA molecules demonstrate that the problem of molecular instability to forces exerted by the probe tip, inherent with scanning probe microscopes, can be prevented.

DNA, Bacterial↗

PubMed1992

Electrostatic spraying of DNA molecules for investigation by scanning tunneling microscopy.

We have investigated electrostatic spraying of DNA onto gold surfaces as an alternative sample-preparation technique for STM studies. Preliminary results show that a higher distribution of isolated strands as well as well ordered aggregates can be obtained with this technique when compared with electrodeposition or drop evaporation. In many places, the well ordered aggregates were found to cleave in a direction perpendicular to their length after repeated scanning in the same direction.

DNA↗

PubMed1992

Scanning tunneling microscopy of DNA: a novel technique using radiolabeled DNA to evaluate chemically mediated attachment of DNA to surfaces.

pBS+ plasmid deoxyribonucleic acid (DNA) was imaged by scanning tunneling microscopy (STM) after mounting microdroplets by aerosol deposition onto heated epitaxial gold surfaces. However, the instability of the adsorbate to forces exerted by the tunneling tip points out the need for more aggressive bonding of molecules to surfaces. We describe a sensitive assay for the qualitative and quantitative evaluation of chemical agents to influence binding of DNA to surfaces using 32P-labeled pBS+ plasmid DNA. We propose that such an assay can make an important contribution to immobilization techniques prior to STM imaging.

DNA↗

PubMed1992

Imaging isolated strands of DNA molecules by atomic force microscopy.

We have employed an atomic force microscope (AFM) to image in air isolated strands of pBS+ plasmid DNA adsorbed onto freshly cleaved mica. At a DNA concentration below 0.3 micrograms/ml isolated strands of the plasmid DNA are usually seen, while for concentrations higher than 3 micrograms/ml a uniform coverage of interconnected DNA strands was observed. We found that the contrast and the width of DNA were dependent upon humidity. When the relative humidity exceeds 60%, negative contrast images with strand widths 20 times the width of DNA are found, while positive contrast images with 7 to 10 times the width of DNA are found when the humidity is below 30%. By placing the AFM in an environment where the humidity could be controlled, we were able to switch between positive and negative contrasts.

Adsorption↗

PubMed1992

Atomic force microscopy of DNA on mica and chemically modified mica.

Atomic force microscopy (AFM) was used to image circular DNA adsorbed on freshly cleaved mica and mica chemically modified with Mg(II), Co(II), La(III), and Zr(IV). Images obtained on unmodified mica show coiling of DNA due to forces involved during the drying process. The coiling or super twisting appeared to be right handed and the extent of super twisting could be controlled by the drying conditions. Images of DNA observed on chemically modified surfaces show isolated open circular DNA that is free from super twisting, presumably due to strong binding of DNA on chemically modified surfaces.

Adsorption↗

PubMed1991

Electrochemically induced adsorption of radio-labeled DNA on gold and HOPG substrates for STM investigations.

In a scanning tunneling microscope (STM) electrochemical cell we have studied the effects of electrode potential on both the surface topography and the adsorption of deoxyribonucleic acid (DNA) to graphite and gold surfaces. Images of the surface of highly oriented pyrolytic graphite (HOPG), of the same area, in response to a positive increase in surface potential show degradation of the step edges with little change in the crystal plane. Images of the same area of a gold surface demonstrate the formation of and the progressive increase in nodular structures on the crystal planes, in response to increased potential, with little effect on the step edges. Using radio-labeled DNA we monitored electrochemical absorption onto HOPG and gold surfaces. Although at no applied potential and at negative surface potentials some DNA was bound, at positive potentials 3 to 5 times more DNA was incorporated onto both surfaces. DNA adsorbed to a surface at a positive potential was not removed by reversing the potential.

Adsorption↗

PubMed1990

Scanning tunneling microscopy and spectroscopy of plasmid DNA.

We present scanning tunneling microscope (STM) images of uncoated deoxyribonucleic acid (DNA) electrochemically mounted on highly-oriented pyrolytic graphite (HOPG) and imaged in air. Images of linear abnormalities inherent to HOPG surfaces that can be confused with DNA are also presented. Scanning tunneling spectroscopic (STS) images generated by superimposing a small, high frequency ac bias onto the dc tunnel bias and recording the ac current signal were taken simultaneously with the topographic images. These spectroscopic images reveal contrast due to local conductivity variations and can be used to differentiate DNA molecules from graphite artifacts.

Carbon↗