Mapping Individual Cosmid DNAs by Direct AFM Imaging

Allison, David P.; Kerper, Peggy S.; Doktycz, Mitchel J.; Thundat, Thomas; Modrich, Paul; Larimer, Frank W.; Johnson, Dabney K.; Hoyt, Peter R.; Mucenski, Michael L.; Warmack, R. J.

doi:10.1006/geno.1997.4686

Cited by 42 publications

(21 citation statements)

References 27 publications

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“…Additionally, we encountered evidence of joining or pairing of CEN DNA molecules by the CBF3 proteins. AFM has been successfully used to image biological samples in air or in liquid (4)(5)(6)(7)(8), and our findings further demonstrate that this imaging method is a powerful tool for the study of macromolecular assemblies and their interactions (9)(10)(11)(12)(13)(14)(15)(16).…”

mentioning

confidence: 52%

Probing the Saccharomyces cerevisiae centromeric DNA ( CEN DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Pietrasanta

Thrower

Hsieh

et al. 1999

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

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Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy. Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is Ϸ9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent Ϸ55°at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.Accurate chromosome segregation in mitosis and meiosis depends on the correct assembly of kinetochores on centromeric DNA (CEN DNA). During cell division, these structures attach chromosomes to the microtubules of the mitotic spindle and move the replicated chromosomes to opposing spindle poles.The minimal functional centromere in the budding yeast Saccharomyces cerevisiae contains a 125-base-pair sequence (CEN) present once on each chromosome, which is organized into three domains: CDEI, CDEII, and CDEIII ( In this paper, we describe the use of atomic force microscopy (AFM; also called scanning force microscopy) to probe the organization of the CBF3-CEN DNA complexes. We find that binding of the CBF3 proteins is associated with DNA shortening and bending. The location of the center of the CBF3 complex relative to the CDEIII site was found to be quite asymmetric, corroborating previous findings from DNase protection and DNA-protein crosslinking studies (2, 3). Additionally, we encountered evidence of joining or pairing of CEN DNA molecules by the CBF3 proteins. AFM has been successfully used to image biological samples in air or in liquid (4-8), and our findings further demonstrate that this imaging method is a powerful tool for the study of macromolecular assemblies and their interactions (9-16). MATERIALS AND METHODSPreparation of CBF3 Proteins. Cbf3a, Cbf3b, and Cbf3c͞d complex proteins tagged with 6-10 histidine residues were overexpressed and purified from Pichia pastoris, Escherichia coli, and S. cerevisiae, respectively, according to the procedure described in ref. 23.Preparation of CEN DNA Fragments and pUC19 DNA Beads. A linear DNA restriction fragment con...

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mentioning

confidence: 52%

Probing the Saccharomyces cerevisiae centromeric DNA ( CEN DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Pietrasanta

Thrower

Hsieh

et al. 1999

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

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“…In spite of its simplicity, backbone contour length appears remarkably accurate for the molecule sizes tested (∼300-20 000 bp) [167,168,[186][187][188][189]. While the conditions varied among studies, single measurement sizing accuracy (defined here as population CV) better than ±2-5% was reported for most cases for distances larger than 1000 bp.…”

Section: Mathematical Analysismentioning

confidence: 96%

“…Using the latter method, Allison et al reported in 1996 [166] and 1997 [167] accurate, AFM-based EcoRI maps of large molecules, plasmids ranging from 3200 to 6800 bp, a cosmid vector (35 000 bp) and the lambda phage genome (48 000 bp). Importantly, they used a special mutant version of EcoRI, obtained from Modrich [190], that binds with reasonably high affinity to its recognition sites, but does not cut.…”

Section: Experimental Systems For High Density Ordered Restriction Mamentioning

confidence: 99%

Single molecule transcription profiling with AFM

et al. 2006

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Established techniques for global gene expression profiling, such as microarrays, face fundamental sensitivity constraints. Due to greatly increasing interest in examining minute samples from microdissected tissues, including single cells, unorthodox approaches, including molecular nanotechnologies, are being explored in this application. Here, we examine the use of single molecule, ordered restriction mapping, combined with AFM, to measure gene transcription levels from very low abundance samples. We frame the problem mathematically, using coding theory, and present an analysis of the critical error sources that may serve as a guide to designing future studies. We follow with experiments detailing the construction of high density, single molecule, ordered restriction maps from plasmids and from cDNA molecules, using two different enzymes, a result not previously reported. We discuss these results in the context of our calculations.

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“…Previous studies [21,22] have shown that sequence-specific topographic labelling of DNA is possible, but the labelling chemistries used could not produce tags with the needed frequency and fidelity for recognizing large numbers of cDNA-sized nucleic acid species in a mixture, as do the nicking enzymes used here. Nicking enzymes have been used previously with fluorescent labelling to map sequences on single DNA molecules using optical microscopy [23,24].…”

Section: Discussionmentioning

confidence: 94%

“…for 30 s. Samples are imaged in tapping mode in air with the Dimension ICON AFM (Bruker Metrology) using k ¼ approximately 3 N m 21 silicon probes (Nanosensors). Image resolution was 2 nm pixel 21 . DNA contour lengths and streptavidin label locations were measured manually with NIH IMAGEJ.…”

Section: Atomic Force Microscopy Imagingmentioning

confidence: 99%

Identifying individual DNA species in a complex mixture by precisely measuring the spacing between nicking restriction enzymes with atomic force microscope

Reed

Hsueh²,

Lam³

et al. 2012

J. R. Soc. Interface.

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We discuss a novel atomic force microscope-based method for identifying individual short DNA molecules (,5000 bp) within a complex mixture by measuring the intra-molecular spacing of a few sequence-specific topographical labels in each molecule. Using this method, we accurately determined the relative abundance of individual DNA species in a 15-species mixture, with fewer than 100 copies per species sampled. To assess the scalability of our approach, we conducted a computer simulation, with realistic parameters, of the hypothetical problem of detecting abundance changes in individual gene transcripts between two single-cell human messenger RNA samples, each containing roughly 9000 species. We found that this approach can distinguish transcript species abundance changes accurately in most cases, including transcript isoforms which would be challenging to quantitate with traditional methods. Given its sensitivity and procedural simplicity, our approach could be used to identify transcript-derived complementary DNAs, where it would have substantial technical and practical advantages versus established techniques in situations where sample material is scarce.

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Mapping Individual Cosmid DNAs by Direct AFM Imaging

Cited by 42 publications

References 27 publications

Probing the Saccharomyces cerevisiae centromeric DNA ( CEN DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Probing the Saccharomyces cerevisiae centromeric DNA ( CEN DNA)–binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy

Single molecule transcription profiling with AFM

Identifying individual DNA species in a complex mixture by precisely measuring the spacing between nicking restriction enzymes with atomic force microscope

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