&lt;title&gt;Atomic force microscopy of DNA-colloidal gold and DNA-protein complexes&lt;/title&gt;

Niu, Luming; Shaiu, Wen‐Ling; Vesenka, James; Larson, Drena D.; Henderson, Eric

doi:10.1117/12.146706

Cited by 4 publications

(2 citation statements)

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“…In addition to the +/− screening and sizing applications discussed here, AFM can be used to visualize DNA molecules that have been allowed to interact with proteins, in which the proteins recognize specific short stretches of sequence within the DNA molecule, such as restriction enzymes. ,, By directly visualizing the position of the binding proteins, the distance between the binding sites can be determined, and the order of the fragments is maintained. This is a significant advantage over gel electrophoresis of restriction digests, where the order of fragments is lost.…”

Section: Discussionmentioning

confidence: 99%

Solid-State DNA Sizing by Atomic Force Microscopy

Spisz

Wiltshire

et al. 1998

Anal. Chem.

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Atomic force microscopy (AFM) allows rapid, accurate, and reproducible visualization of DNA adsorbed onto solid supports. The images reflect the lengths of the DNA molecules in the sample. Here we propose a solid-state DNA sizing (SSDS) method based on AFM as an analytical method for high-throughput applications such as finger-printing, restriction mapping, +/- screening, and genotyping. For this process, the sample is first deposited onto a solid support by adsorption from solution. It is then dried and imaged under ambient conditions by AFM. The resulting images are subjected to automated determination of the lengths of the DNA molecules on the surface. The result is a histogram of sizes that is similar to densitometric scans of DNA samples separated on gels. A direct comparison of SSDS with agarose gel electrophoresis for +/- screening shows that it produces equivalent results. Advantages of SSDS include reduced sample size (i.e., lower reagent costs), rapid analysis of single samples, and potential for full automation using available technology. The high sensitivity of the method also allows the number of polymerase chain reaction cycles to be reduced to 15 or less. Because the high signal-to-noise ratio of the AFM allows for direct visualization of DNA-binding proteins, different DNA conformations, restriction enzymes, and other DNA modifications, there is potential for dramatically improving the information content in this type of analysis.

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Section: Discussionmentioning

confidence: 99%

Solid-State DNA Sizing by Atomic Force Microscopy

Spisz

Wiltshire

et al. 1998

Anal. Chem.

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“…Enzymes such as RNA polymerase (2-4), wild-type EcoRI endonuclease (5), and other proteins (6) bound to short pieces of DNA have been imaged by the atomic force microscope (AFM). Also, a site-specific antibody for Z-DNA sequences bound to a plasmid has been imaged (7).…”

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confidence: 99%

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

Allison

Kerper

Doktycz

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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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 104. ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-.8-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.Restriction endonucleases are enzymes that recognize and bind to specific nucleotide sequences resulting in cleavage of the DNA molecule. The frequency with which these enzymes cleave DNA is determined by both the number and order of nucleotides in the recognition site. Since each position in a specific sequence can be occupied by one of four possible nucleotides, an endonuclease that recognizes a specific sequence of 3 nt would be expected to cleave DNA every 64 bases while an enzyme that is site specific for a 6-nt sequence would cleave DNA, on the average, every 4096 bases. Because restriction enzymes are site specific, their attachment sites can serve as physical map points along DNA molecules.Conventional methods for locating the restriction sites, for a specific endonuclease, rely on cutting the DNA molecules into fragments. Ordering of the fragments and consequent identification of the restriction sites is typically accomplished by a combination of techniques that include multiple partial endonuclease digestion, gel fractionation, Southern blot analysis, and hybridization with labeled end probes (1). As the number of restriction sites to be identified and, therefore, the molecular length of the DNA increases, ordering the restriction sites becomes more difficult. For example, locating the expected 8-15 EcoRI or BamHI sites on a 100-kb cloned DNA would require roughly a month's work and may be compromised by the presence of rapidly cleaved recognition sites.An alternative to identifying restriction sites by fragment analysis would be to use microscopy to image restriction sites on intact DNA molecules. Enzymes such as RNA polymerase (2-4), wild-type EcoRI endonuclease (5), and other proteins (6) bound to short pieces of DNA have been imaged by the atomic force microscope (AFM). Also, a site-specific antibody for Z-DNA sequences bound to a plasmid has been imaged (7). However, imaging the relatively small EcoRI endonuclease site specifically bound to long DNA molecules is more challenging due to the small size of the protein that may be confused with background noise, especially when large areas are imaged.EcoRI is a dimeric globular protein of known sequence with a molecular weight of 62,000. The nucleic acid recognition site is GA...

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