A study of parameters that influence the HPLC and CE separation of double stranded DNA fragments and DNA mutants

The present study evaluates HPLC and CE experimental parameters, such as column packing properties, mobile phase pH, column temperature, and gel type on the separation of DNA fragments and heteroduplexes. The results of this study show that both HPLC and CE are useful techniques for the separation of DNA fragments and for the detection of DNA mutants. Not all HPLC columns tested resolved the DNA fragments or detected the mutation. The packing material type and physical and chemical properties play a significant role. It was found that ion-pair DHPLC is easier and faster than single capillary CE for detecting DNA mutants at their melting temperature (5 min. vs 25 min.). However, CE is faster for the separation of DNA fragments (6.5 min. vs 20 min.). Most HPLC column packing materials tested resolved small DNA fragments, less than 200 bp, but not large fragments, greater than 200 bp. DNA fragments, 21-587 bp, are resolved by DHPLC at 40-50 degreesC, but not at room temperature, using special columns; ultra pure, large pore, high coverage C-18-silica, nonporous C-14-silica or nonporous polystyrene/divinylbenzene and ion-pair gradient elution. CE can resolve the 21-587 bp fragments at room temperature, and at 30 to 50 degreesC. It was also found that fluorocarbon coated capillary filled with polyacryl amide or hydroxycellulose liquid polymers, was more stable than DB-17, however, both capillaries gave good and reproducible CE results.

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