title: Atomic Force Microscope nanolithography on chromosomes to generate single-cell genetic probes.
abstract: BACKGROUND
  Chromosomal 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Atomic 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.
authors.affiliation: Department of Basic and Applied Biology, University of L'Aquila, Via Vetoio 1, L'Aquila 67100, Italy. sebastiano.dibucchianico@cc.univaq.it
authors.forename: Sebastiano
authors.initials: S
authors.lastname: Di Bucchianico
authors.forename: Anna M
authors.initials: AM
authors.lastname: Poma
authors.forename: Maria F
authors.initials: MF
authors.lastname: Giardi
authors.forename: Luana
authors.initials: L
authors.lastname: Di Leandro
authors.forename: Francesco
authors.initials: F
authors.lastname: Valle
authors.forename: Fabio
authors.initials: F
authors.lastname: Biscarini
authors.forename: Dario
authors.initials: D
authors.lastname: Botti
mesh_terms: D000818:Animals; D001717:Birds; D002875:Chromosomes / Q000737:chemistry*; D015342:DNA Probes / Q000737:chemistry*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D042282:Microdissection / Q000379:methods; D018625:Microscopy, Atomic Force; D036103:Nanotechnology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D015342:DNA Probes