Introduction: Although genome sequencings of several model organisms have completed, many plants and animals with a little or no genome sequence still remain, because the whole genome shotgun sequencing which is a main tool in genome sequencing spends huge time and expense. So far, we have been developing a novel method for genome analysis using scanning probe microscope, and established scratch method to recover a nanometer-size chromosome fragment by AFM [1, 2]. Recovery success rate with this method were 90 % or more, and average recovery width is ca. 250nm (equivalent of 500 kbp in silkworm chromosome). However, DNA content with a few Mbp was required for DNA amplification. So, we improved the dissection and recovery method of the chromosome fragments to enlarge the recovery width.
Experimental: AFM (NanoWizard, JPK) with a closed-loop scanner to enable an accurate dissection position of chromosome was used. Silkworm pachytene chromosomes were obtained from oocytes or testes of silkworm fifth instar larvae, and prepared on slide or cover glasses. AFM was equipped with an inverted optical microscope to search the target chromosomes easily. The alternating contact mode was used in AFM measurements and recovery of chromosome fragments.
Results and Discussion: It was found that swollen chromosome fragments were readily recovered [1]. So, we tried to recover chromosome fragments in water, which cause further swelling of the chromosome. As a result, recovery width was increased to about 600 nm, which was two times as wide as average recovery width in air. Furthermore, we have achieved recovered width of 760 nm by continuously three times recovery using the same single AFM-tip at specific region in air without swelling effect. This width is speculated to correspond to 1.5 Mbp genome contents of silkworm pachytene chromosome. This method will be a useful tool for the genome analysis in specific region of a chromosome and has a possibility to contribute for the identification of valuable genes. This research was supported by a fund from the Bio-Oriented Technology Research Advancement Institution (BRAIN).
[1] K. Tsukamoto et al, Nanotechnology, 17, 1391(2006)
[2] K. Tsukamoto et al, Jpn. J. Appl. Phys., 45(3B), 2337(2006) |