Comparison of three methods for measuring locations of long range base pairing in single-stranded DNA by electron...

A comparison was made of three procedures used for measuring the locations of long range base pairing in single-stranded DNA. All three procedures gave equivalent results for the location of a transposon, Tn903, and for a loop created by pairing between the nut and operator segments on an EcoRI restriction fragment of lambda DNA. DNA molecules prepared by a T4 gene 32 protein procedure had the best contrast. The highest frequency of measurable loops resulted from a procedure which utilized ammonium acetate/formamide spreading conditions. This procedure also appeared to be the simplest and most satisfactory procedure. Only one procedure revealed the fact that the nutR and oR regions are separated by several hundred bases, but this procedure, which involves crosslinking with trioxsalen, gave generally unsatisfactory DNA morphology. None of the procedures revealed the existence of a short "bulge" loop of single-stranded DNA located between nutL and oL and a longer "bulge" loop located between nutR and oR.