Simultaneous Detection of Near-field Topographic and Fluorescence Images of Human Chromosomes Via Scanning Near-field Optical/Atomic-force Microscopy (SNOAM)

Iwabuchi, Shinichiro; Muramatsu, Hiroshi; Chiba, Norio; Kinjo, Yasuhito; Murakami, Yuji; Sakaguchi, Toshifumi; Yokoyama, Kenji; Tamiya, Eiichi

doi:10.1093/nar/25.8.1662

Cited by 28 publications

(17 citation statements)

References 7 publications

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“…) images obtained by this method can visualize differences in surface stiffness or local elasticity of single human chromosomes (Nomura et al 2005;Kawabata et al 2007). On the other hand, AFM combined with scanning near-field optical microscopy (SNOM), or SNOM/AFM is one of the candidates for an attractive tool for the study of chromosomes, because it can collect both topographic and fluorescence images of the same portion of the samples simultaneously (Iwabuchi et al 1997;Yoshino et al 2002. Thus, SNOM/AFM is expected to be used for future studies of the structure of chromosomes in relation to the localization of their components labeled with fluorescent substances.…”

Section: Discussionmentioning

confidence: 99%

Atomic force microscopy for imaging human metaphase chromosomes

Ushiki

Hoshi

2008

Chromosome Res

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The present study introduces the principle of atomic force microscopy (AFM) and reviews our results of human metaphase chromosomes obtained by AFM. AFM imaging of the chromosomes revealed that the chromatid arm was not uniform in structure but had ridges and grooves along its length, which was most prominent in the late metaphase. The arrangement of these ridges and grooves was roughly symmetrical with the counterpart of the paired sister chromatids. AFM imaging of banded chromosomes also showed that the ridges and grooves were related to the G/Q-positive and G/Q-negative bands, respectively. At high magnification, the chromatid was seen to be produced by the compaction of highly twisted chromatin fiber loops, and its compaction tended to be stronger in the ridged regions of the chromosomes than in the grooved regions. Our AFM studies also showed the presence of catenation of chromatin fibers between the ridged portions of the chromatid in the late metaphase. Thus, AFM is useful for obtaining the three-dimensional surface topography not only in ambient conditions but also in physiological liquid conditions, and is expected to be an attractive tool for investigating the structure of chromosomes.

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Section: Discussionmentioning

confidence: 99%

Atomic force microscopy for imaging human metaphase chromosomes

Ushiki

Hoshi

2008

Chromosome Res

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“…The detection of nucleic acids by fluorescent dyes such as SYBR Green I (SG) has become increasingly important for a variety of analytic and diagnostic applications (1). Since its introduction in the early 1990s (2), SG has been applied successfully in the detection of nucleic acids in gels (3)(4)(5)(6), in solution (7,8), in the determination of DNase or telomerase activities (9,10), in fluorescence imaging techniques (11), in flow cytometry (12,13), in real-time PCR (14)(15)(16)(17), in biochip applications (18), and more recently, in the demanding quantification of dsDNA in crude extracts of environmental samples that is usually hampered by a variety of quenching processes (19,20).…”

Section: Introductionmentioning

confidence: 99%

Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications

Zipper

Brunner²,

Bernhagen³

et al. 2004

Nucleic Acids Research

678

454

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The detection of double-stranded (ds) DNA by SYBR Green I (SG) is important in many molecular biology methods including gel electrophoresis, dsDNA quantification in solution and real-time PCR. Biophysical studies at defined dye/base pair ratios (dbprs) were used to determine the structure-property relationships that affect methods applying SG. These studies revealed the occurrence of intercalation, followed by surface binding at dbprs above approximately 0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA)* poly(dT) and poly(dG)* poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least approximately 11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+. These structure-property relationships help in the design of methods that use SG, in particular dsDNA quantification in solution and real-time PCR.

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“…We introduce nanolithography on chromosomes surface. In our laboratory we have reduced the 17 μN applied force for the achievement of hybridization probes used in Iwabuchi's work and co-authors [ 14 ], until 5 μN, minimum value successfully used by us. The applied forces are comparable to those used by Oberringer and co-workers [ 15 ].…”

Section: Discussionmentioning

confidence: 99%

Atomic Force Microscope nanolithography on chromosomes to generate single-cell genetic probes

Bucchianico

Poma

Giardi

et al. 2011

Journal of Nanobiotechnology

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BackgroundChromosomal dissection provides a direct advance for isolating DNA from cytogenetically recognizable region to generate genetic probes for fluorescence in situ hybridization, a technique that became very common in cyto and molecular genetics research and diagnostics. Several reports describing microdissection methods (glass needle or a laser beam) to obtain specific probes from metaphase chromosomes are available. Several limitations are imposed by the traditional methods of dissection as the need for a large number of chromosomes for the production of a probe. In addition, the conventional methods are not suitable for single chromosome analysis, because of the relatively big size of the microneedles. Consequently new dissection techniques are essential for advanced research on chromosomes at the nanoscale level.ResultsWe report the use of Atomic Force Microscope (AFM) as a tool for nanomanipulation of single chromosomes to generate individual cell specific genetic probes. Besides new methods towards a better nanodissection, this work is focused on the combination of molecular and nanomanipulation techniques which enable both nanodissection and amplification of chromosomal and chromatidic DNA. Cross-sectional analysis of the dissected chromosomes reveals 20 nm and 40 nm deep cuts. Isolated single chromosomal regions can be directly amplified and labeled by the Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP-PCR) and subsequently hybridized to chromosomes and interphasic nuclei.ConclusionsAtomic force microscope can be easily used to visualize and to manipulate biological material with high resolution and accuracy. The fluorescence in situ hybridization (FISH) performed with the DOP-PCR products as test probes has been tested succesfully in avian microchromosomes and interphasic nuclei. Chromosome nanolithography, with a resolution beyond the resolution limit of light microscopy, could be useful to the construction of chromosome band libraries and to the molecular cytogenetic mapping related to the investigation of genetic diseases.

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Simultaneous Detection of Near-field Topographic and Fluorescence Images of Human Chromosomes Via Scanning Near-field Optical/Atomic-force Microscopy (SNOAM)

Cited by 28 publications

References 7 publications

Atomic force microscopy for imaging human metaphase chromosomes

Atomic force microscopy for imaging human metaphase chromosomes

Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications

Atomic Force Microscope nanolithography on chromosomes to generate single-cell genetic probes

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