Micropreparative fractionation of DNA fragments on metathesis-based monoliths: influence of stoichiometry on separation.
Applying Grubbs' first generation benzylidene-type catalyst Cl2Ru(PCy3)2(CHPh) in ring opening metathesis polymerization (ROMP) of norborn-2-ene (NBE) and 1,4,5,8,8a-hexahydro-1,4,5,8, exo, endo-dimethanonapthalene (DMN-H6), various monoliths were prepared within the confines of silanized borosilicate columns (100x3 mm I.D.) and investigated for the micropreparative separation of pBR322 DNA-Hae III restriction fragments ranging in size from 51 to 587 base pairs (bp), as a sample of double-stranded (ds) DNA. The approach to good resolution of dsDNA on monolithic columns entailed the modulation of the polymer morphology in terms of structure and porosity to suit such an analysis. Structural variations were achieved by changing the relative ratios of comonomers (NBE+DMN-H6) at the expense of porogens, and by increasing the DMN-H6 to NBE mass ratio. For dsDNA separations, eluents comprised 0.1 M aqueous triethylammonium acetate, pH 7.0, and acetonitrile. Alternatively, methanol was introduced in this study as a less polar gradient former. In terms of column evaluation, each column prepared was first tested in the separation of 5'-phosphorylated oligodeoxythymidylic acids [p(dT)(12-18)], since good separation of oligodeoxynucleotides indicates the potential liability of the column tested for dsDNA analysis, and vice versa. It was noted that monoliths with combinations of 25:25:40:10, 28:28:35:9, and 30:30:32:8 (as weight% of NBE/DMN-H6/2-propanol/toluene) showed good resolution of p(dT)(12-18). Moreover, they demonstrated good separation of the first 12 fragments (51-267 bp) of the pBR322 DNA-Hae III digest; however, reduced resolution in the separation of the last five highest molecular mass fragments (434-587 bp) was experienced. The best separation of these fragments was accomplished on a 25:25:40:10 NBE/DMN-H6/2-propanol/toluene combination at a flow-rate of 2 ml/min, a temperature of 50 degrees C, and a gradient of 4-10% acetonitrile in 1 min, then 10-16% in 14 min. The total amount of pBR322 HaeIII digest that may be fractionated on these systems is 0.5-2.5 microg.
Institute of Analytical Chemistry and Radiochemistry, University of Innsbruck, Austria.
Said
S
Lubbad
Betina
B
Mayr
Christian G
CG
Huber
Michael R
MR
Buchmeiser
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D005990:Glycerol / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
D004247:DNA; D005990:Glycerol