The manipulation of DNA with restriction enzymes in low water systems.
The cleavage of phage lambda (lambda) DNA by the restriction enzyme HindIII in low water systems has been investigated. Two types of low water systems have been studied--those which contain a surfactant in a reverse micelle environment and a surfactant-free system in which a solid support (celite) is used. The effect of the surfactants themselves in a normal aqueous environment has also been studied. Charged surfactants were found to greatly inhibit HindIII activity in aqueous buffer, while non-ionic surfactants did not affect either the activity or the specificity of the restriction enzyme. The rate of cleavage by HindIII in a reverse micelle system consisting of sodium dioctylsulphosuccinate is very slow, however, in a Triton B system the expected fragments are observed. In a surfactant-free low water environment, cleavage occurs at the expected sites but in a different order to that observed in normal aqueous systems. These results suggest that DNA tertiary structure in low water systems is different to that in aqueous solution and that this influences cleavage by the restriction enzyme HindIII.
Hanley
A B
AB
A.F.R.C. Institute of Food Research, Norwich, U.K.
Furniss
C S
CS
Kwiatkowska
C A
CA
Mackie
A R
AR
D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D015247:Deoxyribonuclease HindIII / Q000378:metabolism*; D007692:Diatomaceous Earth; D004143:Dioctyl Sulfosuccinic Acid / Q000737:chemistry; D008823:Micelles; D015183:Restriction Mapping; D012995:Solubility; D013501:Surface-Active Agents / Q000737:chemistry; D014867:Water / Q000737:chemistry
D004279:DNA, Viral; D008823:Micelles; D013501:Surface-Active Agents; D014867:Water; D004143:Dioctyl Sulfosuccinic Acid; D007692:Diatomaceous Earth; D015247:Deoxyribonuclease HindIII