Comparison of behavior of N-substituted acrylamides and celluloses on double-stranded DNA separations by capillary electrophoresis at 25 degrees and 60 degrees C.
The behavior of N-substituted poly(acrylamides) and of cellulose in the separation of double-stranded DNA by capillary zone electrophoresis (CZE) is evaluated. Contrary to previously held belief, which attributed best separations in poly(acrylamides) to small DNA fragments (typically in the 50-1000 bp size range) and in celluloses to large DNA fragments, we show that also celluloses can achieve fine sieving of short DNA sizes provided they are used at much higher concentrations than previously reported. For example, in the case of hydroxyethylcellulose (HEC), typically used at 0.2-0.8% concentrations, levels of 3% produce excellent patterns, at 25 degrees C, in the 50-600 bp size range. If separations are conducted at 60 degrees C, sieving is lost in most liquid polymers. However, if the concentration of HEC is raised to 6% and that of hydroxypropylmethylcellulose (HPMC) above 1%, sieving is fully restored. Also, N-substituted acrylamido derivatives, notably N-acryloylaminoethoxyethanol (AAEE) and N-acryloylaminopropanol (AAP) offer excellent performance at both 25 degrees and 60 degrees C, whereas plain poly(acrylamide) irreversibly collapses at high temperatures, possibly due to intrinsic instability of the amido bond under such harsh conditions. Among all the polymers investigated, an 8% solution of poly(AAP) offers the best performance and highest theoretical plates in the 25 degree-60 degree C interval. Separations at high temperatures are necessary when dealing with detection of point mutations in temperature-programmed CZE and are preferred in DNA sequencing. Two additional advantages are evident when running DNA separations at 60 degrees C: a marked reduction of analysis times and a linearization of the transit times of the larger (434-587 bp) DNA fragments, in all polymers tested.
Gelfi
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ITBA, CNR, Milano, Italy.
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D000178:Acrylamides / Q000737:chemistry*; D002482:Cellulose / Q000737:chemistry*; D004247:DNA / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels; D013696:Temperature
D000178:Acrylamides; D005782:Gels; D002482:Cellulose; D004247:DNA