Restriction mapping of recombinant lambda DNA molecules using pulsed field gel electrophoresis.
We have integrated pulsed field gel electrophoresis with the partial digestion strategy of Smith and Birnstiel (1976, Nucleic Acids Res. 3,2387-2398) to generate a rapid and accurate method of restriction endonuclease mapping recombinant lambda DNA molecules. Use of pulsed field gels dramatically improves the accuracy of size determination and resolution of DNA restriction fragments relative to standard agarose gels. Briefly, DNA is partially digested with restriction enzymes to varying extents and then hybridized with a radiolabeled oligonucleotide which anneals specifically to one of the lambda cohesive (cos) ends, effectively end labeling only those digestion products containing that cos end. In this study, we have used an oligonucleotide hybridizing to the right cos end. DNA is then fractionated by pulsed field gel electrophoresis, the gel dried down, and cos end containing fragments visualized by autoradiography. Fragment sizes indicate the distances from the labeled cos end to each restriction site for the particular restriction enzyme employed. This procedure requires only minimal quantities of DNA and is applicable to all vectors utilizing lambda cos ends.
Goode
B L
BL
Department of Biological Sciences, University of California, Santa Barbara 93106.
Feinstein
S C
SC
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004274:DNA, Recombinant / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis; D004586:Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel; D008969:Molecular Sequence Data; D015183:Restriction Mapping
D004274:DNA, Recombinant; D004279:DNA, Viral