Fluidified polyacrylamides as molecular sieves in capillary zone electrophoresis of DNA fragments

Abstract

In order to optimize the separation of DNA fragments, in the 50–500 base pairs (bp) range, a typical size interval of most polymerase chain reaction-amplified DNA chains produced for analysis of genetic diseases, different ways of preparing liquid linear polyacrylamides were evaluated. Standard linear polyacrylamides (PAA), as prepared with typical levels of catalysts (1 μl of pure N,N,N′,N′-tetramethylethylenediamine and 4 μl of 10% peroxodisulfate per ml of gelling solution) at room temperature, have extremely high weight-average molecular mass ( M w) values (in excess of 2 · 10 6) and can be injected or extruded from a capillary at concentrations above 6% only with great difficulty. The same polyacrylamide, if subjected to mastication by ultrasound at 45 kHz for up to ca. 50 h, exhibits much reduced viscosities (e.g. 700 vs. 3600 mPa s, at 8% concentration) and chain lengths ( M w ca. 550 000) and offers increased resolution in the 50–500 bp interval. However, chain rupture by ultrasound produces charged chains, which migrate out of the capillary under the influence of an electric field, thus impairing resolution. Two other ways have been found to produce uncharged, short chains of very low viscosity: chain termination in 2-propanol by polymerization at 35 and 70°C, respectively. The latter process produces chains of M w as low as 230 000 ( M n = 55 000; polydispersity = 4.2) with a viscosity of only 350 mPa s for a 10% polymer solution. In the separation of the seventeen DNA fragments of the marker pBR322/HaeIII (ranging in size from 51 to 587 bp), a 6% solution of “short-chain polyacrylamide (PAA)” affords a resolution of 880 000 theoretical plates, vs. 440 000 for “long-chain PAA”. In a biological sample of a multiplex Duchenne muscular dystrophy containing eighteen DNA fragments, “short-chain PAA” resolves 17 of them, compared with a pattern of only eleven zones in “long-chain PAA”.