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

Abstract

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.

Detection of human erythrocytes influenced by iron deficiency anemia and thalassemia using atomic force microscopy

“…This allows for the observation of the specimen in conditions close to its natural environment. AFM has been used by several research groups to investigate chromosomes (Hoshi and Ushiki, 2011), cell membranes (Suresh and Edwardson, 2010), proteins (Engel, 2011), DNA (Di Bucchianico et al, 2011), RNA structure (Heus et al, 2011), nucleic acid-protein complexes (Miklaszewska et al, 2004), ligand-receptor binding (Odorico et al, 2007), carbohydrates (Lesoil et al, 2010), lipids, living cells. The unique combination of high-resolution imaging and functionality in a physiological environment has made AFM useful for investigating living cells, including yeasts (Moreno-Herrero et al, 2001), bacteria (Lower, 2011), virally infected cells (Chen et al, 2011), and neurons (Ricci et al, 2011).…”

Atomic force microscopy on chromosomes, chromatin and DNA: A review

“…A later innovation was described by Yamanaka et al in 2008 when they used a modified AFM tip as mechanical tweezers to bot cut and 'pick up' a micro dissected part of a metaphase chromosome (Yamanaka et al, 2008). A beautiful example of the power and elegance of AFM micro dissection is described by Di Bucchianico et al (2011) who produced chromosome specific painting probes for chicken micro chromosomes that otherwise would be impossible to differentiate (Di Bucchianico et al, 2011).…”

Correlative nanoscopy: A multimodal approach to molecular resolution

“…AFM itself is a nano‐manipulator. It can push on a particular position of the sample with a very well defined force, move nanosized objects, stimulate mechanically a living cell, or modify a biological specimen (Di Bucchianico et al, 2011 ). All these operations are very time‐consuming and sometimes invasive if the manipulation path is defined from a previously acquired AFM image.…”