RAPID AND ACCURATE SIZING OF DNA FRAGMENTS BY ION-PAIR CHROMATOGRAPHY ON ALKYLATED NONPOROUS POLY(STYRENE-DIVINYLBENZENE) PARTICLES

Ion-pair reversed-phase high-performance liquid chromatography on nonporous alkylated poly(styrene-divinylbenzene) particles allowed the separation of double-stranded DNA fragments in a few minutes with a resolution comparable to that obtained in slab or capillary gel electrophoresis, Using gradients of acetonitrile in 0.1 M triethylammonium acetate, DNA fragments differing in length only by 2-3% could be resolved. Synthesis of the stationary phase was very reproducible, and equilibration of a freshly packed column for at least 2 h proved to be necessary for optimum column performance, whereas column regeneration for 1-5 min was adequate between gradient runs, Various DNA molecular weight markers ranging from 51 to 2176 base pairs in length were used to study the size dependence of retention, A plot of capacity factors versus logarithm of molecular weights revealed a clear correlation between size and retention, Both a local and a global approximation method, which differed with regard to the number of known standard fragments used below and above the fragments with unknown length to create a sizing curve based on simple linear regression, gave essentially identical results, with the inaccuracy of length measurement being 0.05-3.2%. This compares favorably with the accuracy obtained in gel electrophoresis.