www.BioTechniques.com 1041 Vol. 47 | No. 6 | 2009 Benchmarks Many molecular biology techniques require puriication of cDNAs, DNAs, or DNA-RNA hybrids, and/or fractionation of nucleic acids from various biological sources and reactions to improve downstream performance. Generally, PCR amplicons with short primers (~20 bp) and no artifacts can be readily puriied with commercially available PCR puriication kits that have a maximum cutof of 100 bp. However, in multi-step techniques requiring PCR, these puriication kits oten cannot remove artifacts longer than 100 bp; this is especially problematic when DNA libraries must be prepared (1–3). Next-generation low-cell sequencers ofer DNA sequencing directly from PCR, which is advantageous because subcloning is avoided, but it also requires the subsequent puriication of the ampliied products to increase the sequencing quality, which is proportional to the purity of the template (4). In cap analysis of gene expression (CAGE) technologies with deep sequencing or deepCAGE (2)—in which PCR is used as a key step to prepare libraries for flow-cell sequencing—sometimes artifacts <150 bp are generated (1,2). In a search for ways to remove these artifacts, we systematically investigated the efect of NaCl concentration on DNA fraction- ation in the presence of cetyltrimethylam- monium bromide (CTAB) and urea (5,6), using a glass iber matrix in a spin column (illustra GFX Column; GE Healthcare, Little Chalfont, Buckinghamshire, UK). he GFX kit exploits a glass iber matrix in conjunction with chaotropic salt to capture nucleic acids (7,8). Here, to better modulate the fractionation of diferently-sized nucleic acids as a substitute for chaotropic salt, we have introduced a CTAB-urea solution to capture the nucleic acids. Selective purii- cation was performed with variable NaCl concentrations. CTAB has been used for decades to extract and purify DNAs from biological sources (5,6,9–11). It has recently been shown that CTAB along with urea can improve the puriication of RNAs by removing polysaccharides and other contaminants (6). We have also found that RNAs shorter than tRNAs are not precip- itated eiciently under certain conditions (6). With these problems in mind, we have explored various options to optimize this puriication method. To set up the methodology, we started with 500 ng 50-bp DNA ladder marker (Invitrogen, Carlsbad, CA, USA) or PCR reaction (5 μL). he DNAs were mixed with 1% CTAB-urea solution (4 M urea, 50 mM Tris-HCl pH 7.0, and 1 mM EDTA pH 8.0). CTAB forms positive micelles in solution with nucleic acids: it interacts with nucleic acids through the phosphates and its quaternary ammonia, and the long chain of the detergent localizes away from the DNA groove to reduce contact with the surrounding water. It does this by forming aggregates and thereby promotes condensation (10,12). We applied this property of CTAB to capture the DNAs in the glass iber. he exact mechanism of this capture is currently uncharac- terized, but several possibilities are that ( i) micelles are absorbed to the glass ilter matrix, (ii) CTAB coats the iber and makes it positively charged, or (iii) the CTAB-DNA complex is simply retained very eiciently to the surface. he inter- action of CTAB and nucleic acids was controlled by adding 5 M NaCl solution to inal concentrations of 0.4–0.6 M. hen, the mixture was shaken briskly and heated at 65°C for 10 min. Following incubation, samples were kept at room temperature for 10 min and applied to the GFX columns. Each column was centrifuged at 16,000× g for 1 min. Flow-through was collected and eluted DNA was recovered by ethanol precipitation for later control electropho- resis. To remove the CTAB, we washed the column with 600 μL wash bufer (60% ethanol, 0.3 M NaCl, 100 mM Tris-HCl pH 7.5, and 5 mM EDTA pH 8.0). he column was washed a second time with 600 μL 80% ethanol, which removes the CTAB while keeping the DNA precipi- tated onto the matrix. he column was replaced with a new 1.5-mL siliconized tube and DNA was eluted with 50 μL nuclease-free H 2 O, then concentrated by ethanol precipitation. DNA obtained in the irst low-through and ina l elution was analyzed on 6% polyacrylamide gel or 1.5% agarose gel. Gels were stained either with ethidium bromide or Gel Star (Lonza, Rockland, ME, USA) and visualized with BioDocIt (UVP, Upland, CA, USA). We estimated how much DNA was recovered by comparison with a fragmented ladder and quanti ied by measuring the concen- tration with a NanoDrop 1000 spectro- photometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). he overall recovery was ~65%; recovery was ~30% for smaller fragments just above the 150-bp cutof, and ~80% for larger fragments (data not shown). To verify the efectiveness and to search out the diferent cutofs, we tested various salt concentrations (Figure 1, A and B). In general, the cutof value increased with increased salt concentration. For instance, with a 0.6 M salt concentration (used later for Tunable fractionation of nucleic acids Md. Salimullah 1 , Sachiko Kato 2 , Mitsuyoshi Murata 2 , Chika Kawazu 2 , Charles Plessy 1 , and Piero Carninci 1,2,3 1 Functional Genomics Technology Team, Omics Science Center (OSC), RIKEN Yokohama Institute, Kanagawa, Japan, 2 Omics Resource Development Unit, Omics Science Center (OSC), RIKEN Yokohama Institute, Kanagawa, Japan, and 3 LSA Technology Development Group, Omics Science Center (OSC), RIKEN Yokohama Institute, Kanagawa, Japan BioTechniques 47:1041-1043 (December 2009) doi 10.2144/000113249 Keywords: selective purification; CTAB; nucleic acids; size fractionation We developed a method for selective puriication of DNA using the cationic detergent, cetyltrimethylammonium bromide (CTAB), accompanied with urea and controlled high-salt (NaCl) concentration. his method is efective for rapid separation of DNA fragments from artifacts such as PCR primer dimers or ligation adapters. he CTAB-associated puriication completely removed the short PCR artifacts and primers, as well as enzymes and buf- fer, while recovering a suicient quantity of amplicons for subsequent experi- ments such as preparation of libraries. his method could also be applied to the fractionation of nucleic acids generated by other types of reactions. Benchmarks