Large scale preparation of positively supercoiled DNA using the archaeal histone HMf.
A technique to prepare relatively large quantities (>/=100 microg) of highly positively supercoiled DNA is reported. This uses a recombinant archaeal histone (rHMfB) to introduce toroidal supercoils, and an inexpensive chicken blood extract to relax unrestrained superhelical tension. Preparation of positively supercoiled pUC19 DNA molecules, >50% of which have linking number changes ranging from+8 to+17, is demonstrated. Advantages include the high degree of positive supercoiling that can be achieved, control over the extent of supercoiling, easy production of relatively large quantities of supercoiled DNA, and low cost.
LaMarr
W A
WA
Division of Toxicology, 56-787, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Sandman
K M
KM
Reeve
J N
JN
Dedon
P C
PC
D000818:Animals; D001105:Archaea / Q000378:metabolism; D002645:Chickens; D004264:DNA Topoisomerases, Type I / Q000097:blood; D004278:DNA, Superhelical / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D006657:Histones / Q000096:biosynthesis / Q000378:metabolism*; D010957:Plasmids / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
D004278:DNA, Superhelical; D006657:Histones; D011994:Recombinant Proteins; C094495:histone HMf; D004264:DNA Topoisomerases, Type I