A protocol for separation and isolation of small and/or large DNA fragments with high yield using CL4B Sepharose.

Here we describe a simple but eﬀective protocol toseparate a small (<50-bp) DNA fragment from a largeone. Our protocol gives reproducible results and quan-titative recovery of both small and large fragments in arelatively pure form. Furthermore,this is a very gentlemethod with fewer steps than procedures that requirebinding or electrophoretic steps.Separation of biomolecules is necessary to permitanalysis of important subpopulations. We have recentlyhad a need to separate and quantitatively recover smallDNA fragments from mixtures in which they are com-mingled with substantially larger DNA fragments. Ourwork involves identifying and analyzing the chemicalstructures of damage induced in DNA by radiation. Insupport of this,we required a quick and gentle protocolthat minimized the potential of creating handling-in-duced chemical changes in potentially unstable dam-aged-DNA target molecules.Obviously the simplest approach to this problem wasto employ a combination of commercially availablehigh-quality DNA puriﬁcation kits that are designed topurify DNA from enzyme reactions. The majority of kit-based systems rely upon DNA binding to a glass matrixin the presence of chaotropic salts. In our hands suchkits worked well for isolation of larger DNA species,although recovery tended to be highly variable,rangingfrom 20 to 60%. Unfortunately,isolation of small DNAfragments using kit-based systems was even less satis-factory. Using kit-based systems,small DNA fragmentswere recovered in solutions containing high concentra-tions of chaotropic salts and other reaction componentsthat must be removed prior to the analysis of the frag-ments. There are a number of methods reported inthe literature for separation and puriﬁcation of DNAfragments [1–4]. However,most of these methods re-quire multiple manipulative steps.In our case,exposureof damaged DNA to multiple manipulations and highsalt concentrations may facilitate non-radiation-inducedchemical changes to the DNA molecule. Similar andeven more extreme conditions,such as temperaturechanges,are associated with isolation of small DNAfragments from PAGE gels. Consequently,kit-basedprocedures and other standard methods for separationand puriﬁcation of small and large DNA molecules werenot useful for our purpose. Therefore,it was necessaryto develop a fast and gentle DNA fragment separationand puriﬁcation protocol that gives a predictable andconsistently high yield of both large and small DNAfragments from a single procedure.The starting material for this method is CL-4BSepharose (molecular cut oﬀ 70,000Da for globularproteins) obtained from Amersham Pharmacia Biotech(Piscataway,NJ). The CL-4B matrix in 10mM Tris–HCl,pH 8.0,1mM EDTA (TE) was deﬁned and storedat 4 C with 20% ethanol. For each separation,750llofthe deﬁned slurry (ratio of settled beads to total volume2:3) was loaded into a microspin column (Bio-Rad,Hercules,CA). The seal was broken and the column wascentrifuged at 740gfor 1min in a ﬁxed-angle (45 ) mi-crocentrifuge rotor using a 2-ml microcentrifuge tube asa carrier. The column was then equilibrated with TE byadding 100ll of the buﬀer to the column and spinningat 740gfor 1min. The ﬂow through was discarded andthe column was transferred to a new 1.5-ml microfugetube. At this step,the column was ready for sampleapplication.To prepare DNA for use in the characterization ofthis protocol,pTC27 plasmid DNA (Fig. 1A) was re-striction-digested with the unique cutters BglII and MbiI