We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.

Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis

A pseudogene structure in 5S DNA of Xenopus laevis

Publisher Summary This chapter discusses the techniques for DNA sequence analysis by primed synthesis Several rapid methods are available for DNA sequence analysis They rely on high resolution electrophoresis on denaturing polyacrylamide gels to resolve oligonucleotides with one common end but varying in length at the other by a single nucleotide The primed synthesis methods make use of the ability of DNA polymerases to synthesize a complementary radioactive copy of a single-stranded DNA template using restriction enzyme generated DNA fragments as primers The chain terminator sequencing procedure is simple, rapid and accurate This method makes use of the 2',3'-dideoxy and the β-D-arabinofuranosyl analogs of the deoxyribonucleoside triphosphates and their incorporation by DNA polymerase I onto the 3'-hydroxyl of an extending transcript The chain termination sequencing method has a number of advantages over the plus and minus and partial ribosubstitution procedures Every nucleotide shows up as a band, even in runs of the same nucleotide

DNA sequence analysis by primed synthesis.

Isolation and sequence analysis of a barley alpha-amylase cDNA clone.

This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylamide gel matrices, DNA interactions with the two gel matrices, and the effect of curvature on the free solution mobility of DNA are also described. The combined results suggest that the anomalously slow mobilities observed for curved DNA molecules in polyacrylamide gels are primarily due to preferential interactions of curved DNAs with the polyacrylamide gel matrix; the restrictive pore size of the matrix is of lesser importance. In free solution, DNA mobilities increase with increasing molecular mass until leveling off at a plateau value of (3.17 +/- 0.01) x 10(-4) cm2/V s in 40 mM Tris-acetate-EDTA buffer at 20 degrees C. Curved DNA molecules migrate anomalously slowly in free solution as well as in polyacrylamide gels, explaining why the Ferguson plots of curved and normal DNAs containing the same number of base pairs extrapolate to different mobilities at zero gel concentration.

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Peter G.N. Jeppesen

Papers

A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels