[Introduction] Present genome analysis tools, such as shot-gun method are suitable for whole genome and large scale random analysis but require a lot of cost and time, and they are considered to be inappropriate for local or point analyses of desired regions on the genomes. Thus, we have developed a novel genomic analysis method, named "sniper method", that can specifically analyze the desired region of the genome. The method is based on the scanning probe microscope (SPM) technology combined with molecular biology techniques. In this paper, we report a recent result of our project: the creation of new artificial bacterial chromosome (BAC) linkage markers on a silkworm linkage map using nucleotide sequence information obtained from a nano-size chromosome fragments dissected by AFM (atomic force microscopy) at specific genomic position.
[Experimental] The structural imaging of the chromosomes and the dissection of nano-size chromosome fragments were performed by atomic force microscopes (AFM; SPA800P, SII; NanoWizard, JPK) in air at room temperature. For the experiments, silkworm chromosomes in pachytene phase were used. To identify precise position of the BACs were detected by scanning near-field optical/atomic force microscope (SNOM/AFM, SNOAM, SII).
[Results] We succeeded the amplification of DNA from the chromosome fragment with a width of approx. 300 nm dissected by AFM (reported in a related poster in detail). The sequencing of the amplification products provides the sequences of coding regions and the bridge-sequence that fills the gap between the sequence contigs. Using the sequence information, we screened a BAC clone and searched a single nucleotide polymorphism (SNP) in the BAC end sequence between two silkworm strains. Then we performed a linkage analysis and successfully created a new linkage marker on the linkage map. Our newly developed technology will be very useful tool to accelerate the present genome analysis.
This research is supported by a fund from Bio-oriented Technology Research Advancement Institution
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