Electrophoretic strand separation of long DNAs with poly (U,G) in agarose gels

Abstract

We have found that binding of poly(U,G) to single-stranded DNA decreases its mobility in 0.3% agarose gels. Differential binding to the complementary strands of denatured duplex DNA provides a simple method for strand separation. The method is shown to work with bacteriophage lambda DNA, adenovirus DNA and mtDNA for Tetrahymena pyriformis. In all cases the strand that binds more poly(U,G) in CsCl gradients also binds more in gels. The separated strands can be directly blotted from the gel onto nitrocellulose filters and used for hybridization experiments.

In vitro synthesis of late bacteriophage phi 29 RNA

“…DNA was digested with EcoRl and complexed with poly(UG) by a modification of the method of Goldbach et al (14). Specifically, 10 p.g of digested DNA in 0.1 ml of 0.1 mM EDTA was mixed with 0.06 ml of partially hydrolyzed poly(UG) at a concentration of I mg/ml, and the mixture was then heated for 3 min at 100°C to denature the DNA and quickly cooled to 0°C.…”

“…This was achieved by . After the addition of Ficoll and bromphenol blue, samples containing denatured fragments complexed with poly(UG) were subjected to electrophoresis on horizontal 0.45% agarose slab gels, using 0.04 M Trisacetate (pH 7.7)-i mM EDTA as the buffer (14).…”

Vertebrate histone gene transcription occurs from both DNA strands

“…Goldbach et al (6) have described the basic method for strand-separation of recombinant DNA within 'phage X. Differential binding of poly r(UG) to the vector DNA (12) enhances separation of the heat-denatured strands in 0.3% agarose gels.…”