Preparation of Multimilligram Quantities of Large, Linear DNA Molecules for Structural Studies  Merlind Muecke, Martin Samuels, Megan Davey, David Jeruzalmi  Structure  Volume 16, Issue 6, Pages 837-841 (June 2008) DOI: 10.1016/j.str.2008.04.008 Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 1 The Self-Primed DNA-Synthesis Protocol (A) The starting molecule is prepared by conventional PCR by using an appropriate pair of primers. The sequence of these primers consists of a short segment of the sequence of interest, flanked by the cohesive end sequence from the genome of phage lambda (cos), along with EcoRV restriction enzyme sites (see text). (B) The self-priming procedure is initiated by incubation of the 20-fold diluted product from stage 1 at 94°C, followed by annealing at 34°C. Hybridization generates a doubly primed molecule annealed on the cohesive end sequences. Other more complicated annealed products can also form. DNA polymerase converts this species into a duplex that contains a tandem repeat of the molecule of interest. In subsequent cycles, amplification of this species over the course of many cycles of melting, annealing, and DNA synthesis produces kilobase length molecules harboring many repeats of the sequence of interest. (C) In stage 3, the products described in (B) are diluted 10-fold and submitted to a mixed primer-based and self-primed amplification. The primer-based amplification utilizes the original primers from stage 1. The purpose of stage 3 is to further amplify the quantity of concatemeric DNA. (D) The DNA molecule of interest is released from the high-molecular weight polymers by restriction enzyme digestion. Structure 2008 16, 837-841DOI: (10.1016/j.str.2008.04.008) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 2 Typical DNA Molecules Prepared by Using Self-Primed Synthesis DNA molecules of varying lengths were prepared, purified as described in the text, radiolabeled with 32P (Sambrook and Russell, 2001), and analyzed by using an 8% native acrylamide gel. We show preparation, by enzymatic self-primed amplification, of several large DNA molecules whose lengths range from 100–300 bp. Lane 1 contains 100 bp size markers as indicated. Lanes 2–4 contain DNA products of the indicated lengths. Structure 2008 16, 837-841DOI: (10.1016/j.str.2008.04.008) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 3 1.5% Agarose Gel Analysis of the Products of the Self-Primed DNA-Synthesis Protocol Lane 1 shows the preparation of the concatemerogenic DNA duplex by conventional PCR (stage 1). The molecule is 172 bp in length (circled, 136 from the desired sequence + 36 from the cos sequence and restriction sites). Lane 2 shows the products after the third stage of the self-primed procedure and contains high-molecular weight DNA species containing many repeats of the sequence of interest. The length of the products (circled) from stage 3 is greater than 48 kbp and fails to enter the gel. Lane 3 shows the final purified product after enzymatic digestion and purification. The size of the molecules associated with this procedure is established by comparison to size markers (lane 4, 100 bp; lane 5, kilobase; lane 6, lambda genomic DNA). The gel is visualized by staining with ethidium bromide. Structure 2008 16, 837-841DOI: (10.1016/j.str.2008.04.008) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 4 Anion-Exchange Chromatography of Enzymatically Synthesized DNA A 195-mer DNA molecule was synthesized as described in the text. A total of 5 ml of the 15 ml of the final reaction volume were loaded onto a 30 ml Source Q column equilibrated in buffer A. The column was washed to allow unbound material to flow through. Purified DNA was eluted with a linear gradient (black curve: conductivity) from 0% to 50% buffer B. Elution was followed by recording the absorbance at 254 nm (blue curve). Chromatography was carried out at 25°C. A total of 4.4 mg purified 192-mer DNA was recovered after chromatography. Structure 2008 16, 837-841DOI: (10.1016/j.str.2008.04.008) Copyright © 2008 Elsevier Ltd Terms and Conditions