Department of Basic and Applied Biology, University of L'Aquila, Via Vetoio 1, L'Aquila 67100, Italy.
J Nanobiotechnology. 2011 Jun 28;9:27. doi: 10.1186/1477-3155-9-27.
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.
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.
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.
染色体显带技术为从细胞遗传学可识别区域中分离 DNA 以生成荧光原位杂交的遗传探针提供了直接进展，该技术在细胞和分子遗传学研究和诊断中变得非常普遍。有几个描述微切割方法（玻璃针或激光束）从中期染色体获得特定探针的报告。传统的切割方法存在一些局限性，例如需要大量染色体才能生产探针。此外，由于微针的相对较大尺寸，常规方法不适合单染色体分析。因此，新的切割技术对于在纳米尺度上对染色体进行高级研究至关重要。
我们报告了使用原子力显微镜（AFM）作为工具，对单个染色体进行纳米操作，以生成单个细胞特异性遗传探针。除了开发更好的纳米切割方法外，这项工作还侧重于分子和纳米操作技术的结合，这些技术既可以进行纳米切割，又可以对染色体和染色单体 DNA 进行扩增。对切割染色体的横截面分析显示出 20nm 和 40nm 的深切口。分离的单个染色体区域可以通过简并寡核苷酸引物聚合酶链反应（DOP-PCR）直接扩增和标记，然后与染色体和间期核杂交。
原子力显微镜可以轻松地用于以高分辨率和精度可视化和操作生物材料。用 DOP-PCR 产物作为测试探针进行的荧光原位杂交（FISH）已在禽类微染色体和间期核中成功测试。分辨率超过光学显微镜分辨率极限的染色体纳米光刻术对于构建染色体带文库以及与遗传疾病研究相关的分子细胞遗传学作图可能是有用的。
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