Agarose entrapment method for the production of SfiI linking library for Theileria parva.
We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic DNA. The method involves the physical separation of the small proportion of plasmids in a plasmid genomic library that are linearized by SfiI digestion, from the bulk of molecules that remain circular, by ordinary electrophoresis through high-percentage gels of SeaPlaque agarose. Following the isolation of linearized molecules, their recircularization, and introduction into Escherichia coli, 55% of recovered plasmids contained inserts of the expected size, and 73% of these had SfiI sites. This represented a 25-fold enrichment of linking clones expected to be present at a frequency of 1/60 in the original library of 4- 6-kb fragments of genomic DNA of the protozoan parasite Theileria parva. This approach is rapid and obviates the need for introduction of a selectable marker. It is uniquely appropriate for linking clones spanning SfiI sites as this enzyme leaves degenerate 3' overhanging ends that preclude the direct ligation into vector sites required by most alternative strategies, but that favor the recircularization reactions used here.
International Laboratory for Research on Animal Diseases, Nairobi, Kenya.
J R
JR
Young
S P
SP
Morzaria
D000818:Animals; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D004270:DNA, Circular; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D015723:Gene Library; D008040:Genetic Linkage; D016797:Theileria parva / Q000235:genetics*
D004270:DNA, Circular; D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific