FLUIDIFIED POLYACRYLAMIDES AS MOLECULAR-SIEVES IN CAPILLARY ZONE ELECTROPHORESIS OF DNA FRAGMENTS

In order to optimize the separation of DNA fragments, in the 50-500 ba se pairs (bp) range, a typical size interval of most polymerase chain reaction-amplified DNA chains produced for analysis of genetic disease s, different ways of preparing liquid linear polyacrylamides were eval uated. Standard linear polyacrylamides (PAA), as prepared with typical levels of catalysts (1 mu l of pure N,N,N',N'-tetramethylethylenediam ine and 4 mu l of 10% peroxodisulfate per mi of gelling solution) at r oom 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. Th e same polyacrylamide, if subjected to mastication by ultrasound at 45 kHz for up to ca. 50 h, exhibits much reduced viscosities (e.g. 700 v s. 3600 mPa s, at 8% concentration) and chain lengths (M(w) ca. 550000 ) 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 impai ring 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 degrees C, respectively. The latter proce ss produces chains of M, as low as 230000 (M(n) = 55000; polydispersit y = 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 pBR32 2/HaeIII (ranging in size from 51 to 587 bp), a 6% solution of ''short -chain polyacrylamide (PAA)'' affords a resolution of 880000 theoretic al plates, vs. 440000 for ''long-chain PAA''. In a biological sample o f a multiplex Duchenne muscular dystrophy containing eighteen DNA frag ments, ''short-chain PAA'' resolves 17 of them, compared with a patter n of only eleven zones in ''long-chain PAA''.