ABSTRACT
We propose the use of a double reciprocal plot of the inflection slope and the concentration of a sieving polymer to compare sieving properties of polymers regardless of their concentration and to permit selection of appropriate sieving materials. Using this plot, the lines extrapolating the experimental values intersect the ordinate at the value corresponding to the inflection slope at the infinite concentration of the sieving polymer and the abscissa at the value characteristic for the given polymer. The latter corresponds to the polymer concentration at which the inflection slope equals half the inflection slope at an infinite polymer concentration. It is inversely proportional to intrinsic viscosity and this makes intrinsic viscosity an important physicochemical constant suitable for selection of new potent sieving polymers.
Subject(s)
Electrophoresis, Capillary/methods , Models, Chemical , Chemical Phenomena , Chemistry, Physical , DNA/isolation & purification , Electrophoresis, Polyacrylamide Gel/methods , Kinetics , Mathematics , Molecular Weight , Oligodeoxyribonucleotides/isolation & purification , Polymers/chemistry , ViscosityABSTRACT
Purification of galactomannans including guaran, tara gum, and locust bean gum is described as well as their use as a sieving matrix in DNA sequencing by capillary electrophoresis (CE). Three methods of galactomannan purification were developed and tested using guaran. The first method is based on hydrolysis of proteins using alkali treatment and precipitation of guaran with acetone. The second method uses ion-exchange resins QAE Sephadex A-25 and SP Sephadex C-25 together with acetone precipitation. The third method is similar to the second one, except that it uses ion-exchange resins based on polystyrene, Source 30Q and Source 30S. Capillary zone electrophoresis of acetonitrile extracts from guaran revealed 4-5 characteristic major peaks and several minor peaks. Guar gum from different suppliers differed in the content of proteins. In purified guaran, protein peaks were detectable only using a 300-fold concentrate of extract. The content of proteins in the guaran purified using the third method was 0.001% m/m as determined by CE. The weight average molecular mass of purified guaran can be as large as 2.2 x 10(6). The purified galactomannans were used as a sieving matrix in DNA sequencing by CE. M13 DNA was sequenced to read lengths of about 600 bases in less than 90 min. Separation efficiencies exceeded 1 million theoretical plates for DNA fragments shorter than about 600 bases.
Subject(s)
Electrophoresis, Capillary/methods , Mannans/chemistry , Acetonitriles , Chromatography, Ion Exchange , DNA/isolation & purification , Dextrans , Electrophoresis, Polyacrylamide Gel , Galactans/chemistry , Galactose/analogs & derivatives , Mannans/isolation & purification , Molecular Weight , Oligodeoxyribonucleotides/isolation & purification , Plant Gums , Polysaccharides/chemistry , Proteins/isolation & purification , Sequence Analysis, DNA/methods , SonicationABSTRACT
We compare the migration behavior of DNA sequencing fragments in hydroxyethyl cellulose (HEC) to the theoretical model of migration in the reptation mode. Good agreement was found for the mobility curve. We derived empirical equations for the relationship between selectivity per base and sieving matrix concentration and between the mobility slope and matrix concentration. We propose the inflection slope, i.e., the slope of the log-log mobility curve at its inflection point, as the quantitative parameter of sieving performance.
