Applied slalom chromatography improved DNA separation by the use of columns developed for reversed-phase chromatography. 1996

J Hirabayashi, and K Kasai
Department of Biological Chemistry, Teikyo University, Sagamiko, Japan.

Improved resolution in slalom chromatography, a novel size-fractionation method discovered recently for relatively large DNA molecules (> 5 kpb), was obtained by using columns generally employed for reversed-phase chromatography: i.e., two types of Capcell-Pak (methyl or phenyl-derivatized 5-microns microbeads), and five types of Hypersil-3 packings (trimethylsilyl, dimethyloctyl, cyanopropyl, octadecyl or phenyl-derivatized 3-microns microbeads). The resolution of 5-15-kbp DNA was significantly improved by employing these columns, though the separation characteristics differed. When Capcell-Pak columns were used with a normal low-salt eluting solvent (10 mM sodium phosphate, pH 6.8, 1 mM EDTA), chromatograms were obtained for lambda/HindIII fragments (a mixture of 0.1, 0.5, 2.0, 2.3, 4.4, 6.6, 9.4 and 23.1-kbp fragments) similar to those obtained previously with Asahipak GS-310 5-microns size-exclusion packings. However, when up to 0.2 M NaCl was added to the solvent, the DNA was increasingly retarded, particularly the 4.4, 6.6 and 9.4-kbp fragments, resulting in improved resolution in the low to middle molecular-mass range. The effect of salt was more significant with Capcell-Pak Phe than C1, although various features characteristic of slalom chromatography were preserved with both columns; i.e., dependency on DNA size, flow-rate, and temperature. This suggests that a mixed mode of separation, that is, slalom mode and hydrophobic-interaction mode, was operating. Although all of the Hypersil-3 packings showed significant adsorption of lambda/HindIII fragments under low-salt conditions, the fragments could be eluted with satisfactory yield and resolution by adding acetonitrile (> 5%) to the solvent. Notably, these Hypersil-3 packings allowed resolution of a 4.4-kbp lambda/HindIII fragment from the flow-through fraction for the first time, possibly due to their small particle size. Thus, various packing materials developed for high-performance liquid chromatography proved to be applicable for slalom chromatography, though the eluting conditions still need to be refined. The results support the concept that slalom chromatography is based on a hydrodynamic phenomenon.

UI MeSH Term Description Entries
D008863 Microspheres Small uniformly-sized spherical particles, of micrometer dimensions, frequently labeled with radioisotopes or various reagents acting as tags or markers. Latex Beads,Latex Particles,Latex Spheres,Microbeads,Bead, Latex,Beads, Latex,Latex Bead,Latex Particle,Latex Sphere,Microbead,Microsphere,Particle, Latex,Particles, Latex,Sphere, Latex,Spheres, Latex
D002411 Cation Exchange Resins High molecular weight insoluble polymers which contain functional anionic groups that are capable of undergoing exchange reactions with cations. Cation Exchange Resin,Cation Exchangers (Resins),Exchange Resin, Cation,Exchange Resins, Cation,Resin, Cation Exchange,Resins, Cation Exchange
D002627 Chemistry, Physical The study of CHEMICAL PHENOMENA and processes in terms of the underlying PHYSICAL PHENOMENA and processes. Physical Chemistry,Chemistries, Physical,Physical Chemistries
D002850 Chromatography, Gel Chromatography on non-ionic gels without regard to the mechanism of solute discrimination. Chromatography, Exclusion,Chromatography, Gel Permeation,Chromatography, Molecular Sieve,Gel Filtration,Gel Filtration Chromatography,Chromatography, Size Exclusion,Exclusion Chromatography,Gel Chromatography,Gel Permeation Chromatography,Molecular Sieve Chromatography,Chromatography, Gel Filtration,Exclusion Chromatography, Size,Filtration Chromatography, Gel,Filtration, Gel,Sieve Chromatography, Molecular,Size Exclusion Chromatography
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004279 DNA, Viral Deoxyribonucleic acid that makes up the genetic material of viruses. Viral DNA
D015247 Deoxyribonuclease HindIII One of the Type II site-specific deoxyribonucleases (EC 3.1.21.4). It recognizes and cleaves the sequence A/AGCTT at the slash. HindIII is from Haemophilus influenzae R(d). Numerous isoschizomers have been identified. EC 3.1.21.-. DNA Restriction Enzyme HindIII,Deoxyribonuclease BstFI,Deoxyribonuclease EcoVIII,Endonuclease HindIII,B Pertussis Restriction Enzyme I,BpeI Endonuclease,Endodeoxyribonuclease BpeI,Endonuclease Asp52I,Endonuclease BbrI,Endonuclease BpeI,Endonuclease BstFI,Endonuclease Cfr32I,Endonuclease ChuI,Endonuclease Eco65I,Endonuclease Eco98I,Endonuclease EcoVIII,Endonuclease Hin1076III,Endonuclease Hin173I,Endonuclease HinJCII,Endonuclease HinbIII,Endonuclease HinfII,Endonuclease HsuI,Endonuclease LlaCI,Endonuclease MkiI,LlaCI, Endonuclease
D055598 Chemical Phenomena The composition, structure, conformation, and properties of atoms and molecules, and their reaction and interaction processes. Chemical Concepts,Chemical Processes,Physical Chemistry Concepts,Physical Chemistry Processes,Physicochemical Concepts,Physicochemical Phenomena,Physicochemical Processes,Chemical Phenomenon,Chemical Process,Physical Chemistry Phenomena,Physical Chemistry Process,Physicochemical Phenomenon,Physicochemical Process,Chemical Concept,Chemistry Process, Physical,Chemistry Processes, Physical,Concept, Chemical,Concept, Physical Chemistry,Concept, Physicochemical,Concepts, Chemical,Concepts, Physical Chemistry,Concepts, Physicochemical,Phenomena, Chemical,Phenomena, Physical Chemistry,Phenomena, Physicochemical,Phenomenon, Chemical,Phenomenon, Physicochemical,Physical Chemistry Concept,Physicochemical Concept,Process, Chemical,Process, Physical Chemistry,Process, Physicochemical,Processes, Chemical,Processes, Physical Chemistry,Processes, Physicochemical

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