Recovery of intact yeast chromosomal DNA from agarose gel plugs using coil-globule transition.
In the current studies, we designed a new approach for sizing and isolating chromosomal DNA using coil-globule transition, which avoids fragmentation of giant DNA due to mechanical stress. Although coil-globule transition is reversible and globular DNA is tolerant to mechanical stress, globular DNA cannot be manipulated by an electric field because of the loss of its negative charges. In our system, however, DNA is extracted from an agarose gel in a coiled state into a solution of PEG, and coil-globule transition is induced by cations generated at the anode. This method achieves buffer exchange without stirring, which is the main cause of mechanical stress. Real-time analysis of T4dc viral DNA molecules revealed that they change immediately from a coiled to a globular form when the cation concentration is sufficiently high. This method was used to prepare yeast chromosomal DNA in a globular state without fragmentation.
Komatsu
Jun
J
Department of Ecological Engineering, Toyohashi University of Technology, Hibarigaoka, Tempaku-cho, Aichi, Japan.
Nakano
Michihiko
M
Kurita
Hirofumi
H
Takashima
Kazunori
K
Katsura
Shinji
S
Mizuno
Akira
A
D000535:Aluminum / Q000494:pharmacology; D015825:Chromosomes, Fungal / Q000235:genetics; D004271:DNA, Fungal / Q000187:drug effects / Q000302:isolation & purification* / Q000648:ultrastructure*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D009690:Nucleic Acid Conformation; D011092:Polyethylene Glycols / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004271:DNA, Fungal; D011092:Polyethylene Glycols; D000535:Aluminum