Protocol

Enzymatic Synthesis of Multi-Milligram Quantities of Large, Linear DNA Molecules for Structural Studies

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA

Corresponding author (dj{at}mcb.harvard.edu)

INTRODUCTION

Structural analyses of large protein-DNA complexes (such as those associated with replication initiation, eukaryotic transcription activation or chromatin remodeling, among others) remain a challenge because of difficulties in obtaining multi-milligram quantities of high-quality preparations of large, linear DNA molecules. This protocol describes a three-stage DNA amplification procedure for making such molecules in amounts that are suitable for structural studies. In the first step, conventional polymerase chain reaction (PCR) using specialized primer sequences is used to prepare a DNA molecule suitable for self-primed DNA synthesis. This molecule, which consists of the sequence of interest flanked by the cohesive end sequences from bacteriophage as well as endonuclease recognition sites, is then submitted to self-primed DNA synthesis in the second step. Amplification produces long polymers of DNA, tens of kilobases in length, which harbor many copies of the sequence of interest. The yield from the second step is increased in the third phase, which consists of another round of amplification. Finally, endonuclease digestion of these polymers, followed by chromatographic purification, yields high-quality preparations of DNA. The molecules produced by this procedure consist of the DNA sequence of interest with three base pairs at either end. This procedure typically yields 400-800 µg of purified DNA per milliliter of amplification reaction.