Notes Molecular Weight and Polydispersity of Calf-Thymus DNA: Static Light-Scattering and Size-Exclusion Chromatography with Dual Detection Bedr ˇich Porsch,* Richard Laga, Jir ˇı ´ Horsky ´, C ˇ estmı ´r Kon ˇa ´k, and Karel Ulbrich Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., 162 06 Prague, Czech Republic Received July 8, 2009 Revised Manuscript Received September 9, 2009 Plausible calf-thymus DNA molecular weight distribution can be obtained by size-exclusion chromatography with dual low- angle light scattering/refractometric detection at sufficiently low flow rate. The distribution extends over three decades of molecular weight and is characterized by weight average molecular weight M w ) 8418000 and polydispersity index M w / M n ) 5.2. After strongly scattering impurities had been removed from the sample using adsorption properties of the 3 μm mixed- cellulose-ester filter membranes, static light-scattering measure- ment in flow injection mode was feasible and gave M w ) 8580000, corroborating the veracity of SEC results. Introduction Calf-thymus (CT) DNA, which has been commercially available for a long time from various vendors at a reasonable price, is widely used in diverse biophysical and biochemical studies (Web of Science has provided 1140 references to CT- DNA during the latest five years). The commercial samples are referred to as “highly polymerized”, “polydisperse”, “fibrous preparation”, “containing low amount of RNA and proteins”, and characterized by water content and UV absorbance per mass. The information about their molecular weight and polydispersity is not available from the suppliers and cannot be obtained from electrophoresis, routinely used for DNA characterization. In the literature, M w of CT-DNA samples appears scarcely (8000000 1,2 and 6000000, 3 that is, the values are in the ultrahigh molecular weight range); quantitative information on CT-DNA polydis- persity is, to the best of our knowledge, missing completely. This poses a major impediment to physicochemical studies utilizing CT-DNA as experimentally accessible quantities (obtained by methods such as static and dynamic light scattering (LS), viscometry, etc.) are related to the molecular weight in different ways and thus difficult to compare because they are affected by the sample polydispersity differently. Recently, we studied 4 formation and transformation of DNA-poly(lysine) complexes as models for gene delivery vectors, using, among other methods, static and dynamic LS, and found the lack of the information on CT-DNA polydispersity rather frustrating. Polydispersity of macromolecular samples is routinely as- sessed by the size-exclusion chromatography (SEC); however, the molecular weight of CT-DNA is considered too high for successful SEC analysis. 1 In our recent SEC studies of ultrahigh molecular weight poly(ethylene oxide) 5 and ultrahigh molecular weight sodium hyaluronate, 6 we found that their SEC separation is substantially disturbed by diverse flow-retardation effects, including slalom chromatography but that the nonbiased mo- lecular weight distribution might be obtained using sufficiently low mobile phase flow rate. To verify that this approach is valid also for CT-DNA, we performed and optimized SEC of CT- DNA. We found that CT-DNA can indeed be analyzed by SEC if low enough flow rate was used. The procedure described in this note can be used also for other polydisperse DNA samples. To verify SEC results and especially to check that no sample is “lost” within the chromatography column, we also determined M w of CT-DNA by static LS experiments in flow-injection mode. The light-scattering signal from solutions of water-soluble polymers prepared from biological sources is generally degraded by the presence of strongly scattering compact impurities. We found that hydrophobic adsorption on a suitable filtration membrane effectively removes these strongly scattering particles from CT-DNA and makes static LS possible. Experimental Section Materials. CT-DNA sodium salt, type 1, “highly polymerized” (Lot No. 091K7030) having 16.9 A 260 units/mg solid was from Sigma. Analytical reagent grade NaCl was obtained from Merck (Darmstadt, Germany) and used without further purification. Water was from a Millipore Milli-Q PLUS UF ultrapure water purification unit (Millipore Corp., Bedford, MA). Methods. Modular SEC chromatograph consisted of a Shimadzu LC-10ADVp pump (Shimadzu Corp., Kyoto, Japan), a vacuum degas- sing unit DEGASYS (Sanwa Tsusho, Ltd., Tokyo, Japan), a Pharmacia injection valve V-7 with 500 μL loop (Pharmacia and Upjohn, Uppsala, Sweden), a Chromatix KMX-6 low-angle light-scattering detector (LDC/Milton Roy, Sunnyvale, CA) and a Waters 2410 differential refractometer (Waters Assoc., Milford, MA) connected through a Black Star (Huntingdon, UK) 2308 A/D converter to an IBM-compatible computer. The separation column was a TSKgel GMPW linear (7.5 × 600 mm) column, particle size 17 μm, (Watrex, Prague, CR). Aqueous sodium chloride (0.1 M) was used as a mobile phase in all experiments. This SEC setup was transformed to a flow-injection static LS system using a Teflon capillary (length 60 cm, inner diameter 0.5 mm) instead of the SEC column and a 10 mL Superloop (Pharmacia & Upjohn, Uppsala, Sweden), which acts as a mobile-phase-driven syringe, was mounted instead of a capillary loop. The injected volume in flow- injection experiments was usually 4 mL. A detailed description of both techniques is given elsewhere. 5,6 0.1% CT-DNA in mobile phase was prepared by gentle shaking for 3 days. This stock solution was diluted to a nominal working concentration of 25 μg/mL for SEC and static LS experiments (3 h gentle mixing). Sample Filtrations. Filtrations prior to injections were performed using a “Genie” programmable syringe pump (Kent Scientific Corpora- tion, Torrington, CT) at a flow rate of 0.25 mL/min. Hydrophilic PVDF and hydrophobic PTFE membrane filters having porosity 1 μm (Puradisc 13 mm, Whatman, Maidstone, U.K.) and MCE (mixed cellulose esters) membranes with a porosity of 1.2 and 3 μm (Millipore) in a 13 mm Teflon holder (Whatman) were used because coiled macromolecules of CT-DNA are too large for commonly used filters * To whom correspondence should be addressed. E-mail: porsch@ imc.cas.cz. Biomacromolecules 2009, 10, 3148–3150 3148 10.1021/bm900768j CCC: $40.75  2009 American Chemical Society Published on Web 10/09/2009