Introduction:
Although the genome sequencings of several model organisms have been finished, many industrially and agriculturally important animals and plants remain to have no or only little genome information, since present genome analyses require large scale-, cost- and labor-consuming experiments. We have been proceeding with the project to develop a novel genome analysis method, which enables a local and region-specific genome analysis. The method is based on scanning probe microscope (SPM) technology, including atomic force microscopy (AFM) and scanning near-field optical/atomic force microscopy (SNOM/AFM), under the combination with molecular biology. Here we report the recent progress of our project.
Experimental:
or the target, we adopted silkworm genome since its whole genome analysis project is just on going. Silkworm chromosome in meiotic pachytene phase was used as an experiment material. A SNOM/AFM instrument (SNOAM, SII) was used for the simultaneous imaging of the chromosome topographies and the fluorescent signals from fluorescent-labeled bacterial artificial chromosome (BAC) clones, of which data were used for BAC mapping. AFM (SPA-800P, SII; NanoWizard, JPK) were used for observation and nano-scale dissection of the chromosomes. SNOM/AFM and AFM were operated in air at room temperature. DNA amplifications were performed using improved DOP-PCR and MDA methods.
Results:
we established the method for dissection and recovery of a nanometer size chromosome fragments (ca. 300 nm wide) by AFM. Furthermore, the nucleotide sequences were successfully amplified using the dissected chromosome fragments as a template. The amplified DNAs were sequenced and identified as originating from silkworm genome. The obtained sequence information could be used for gap filling and marker creation on a linkage map (see also related poster). The high-resolution detection of BAC clone positions on the chromosomes by SNOM/AFM toward the development of a new, direct physical map construction method is also demonstrated. This work was supported by the funds from the Bio-Oriented Technology Research Advancement Institution (BRAIN).
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