Sizing of single globular DNA molecules by using a circular acceleration technique with laser trapping.
We describe a method for in situ sizing individual huge DNA molecules by laser trapping. Single DNA molecules are reversibly transformed, without mechanical fragmentation of fragile huge-sized DNA, from their random coil state into their globular state induced by condensing agents poly(ethylene glycol) and Mg(2+). With the use of a globular DNA molecule folded by condensation, the critical velocity of the circularly accelerated single globular DNA molecule by laser trapping was found to be proportional to the size of the DNA. Yeast, Saccharomyces cerevisiae, chromosome III (285 kbp) was successfully sized (281 +/- 40 kbp) from a calibration curve scaled using lambda, T4, and yeast chromosome VI (48.5, 166, and 385 kbp, respectively). The use of critical velocity as a sizing parameter makes it possible to size single DNA molecules without prior conformational information, i.e., the radius of a single globular huge DNA molecule as a nanoparticle. A sized single globular DNA molecule could be trapped again for subsequent manipulation, such as transportation of it anywhere. We also investigated a possibility of reusing the globular DNA molecules condensed by PEG and Mg(2+) for PCR and found that PCR efficiency was not deteriorated in the presence of the condensation agents.
Hirano
Ken
K
Health Technology Research Center, National Institute of Advanced Industrial Science and Technology, 2217-14, Hayashi-cho, Takamatsu, Kagawa, 761-0395, Japan. hirano-ken@aist.go.jp
Nagata
Hideya
H
Ishido
Tomomi
T
Tanaka
Yoshio
Y
Baba
Yoshinobu
Y
Ishikawa
Mitsuru
M
D017122:Bacteriophage T4 / Q000737:chemistry / Q000235:genetics; D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry*; D004279:DNA, Viral / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D053844:Lasers, Solid-State; D015636:Magnesium Chloride / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D011092:Polyethylene Glycols / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D011137:Polystyrenes / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D012996:Solutions; D014783:Viscosity
D004271:DNA, Fungal; D004279:DNA, Viral; D005456:Fluorescent Dyes; D011137:Polystyrenes; D012996:Solutions; D015636:Magnesium Chloride; D011092:Polyethylene Glycols