Preparation of Multimilligram Quantities of Large, Linear DNA Molecules for Structural Studies

Abstract

We describe a method for preparing large, linear DNA molecules in amounts that are suitable for structural studies. The procedure employs self-primed DNA amplification on a starting molecule that consists of the sequence of interest flanked by the cohesive end sequences from bacteriophage lambda as well as endonuclease recognition sites. Amplification produces long polymers of DNA, tens of kilobases in length, which harbor many copies of the sequence of interest. Endonuclease digestion of these polymers, followed by chromatographic purification, yields high-quality preparations of the DNA molecule of interest. Reliance on the cohesive end sequences to initiate self-primed amplification effectively enables the synthesis of DNA molecules of interest with minimal restriction on length and sequence.

Biophysical Characterization of the Centromere-specific Nucleosome from Budding Yeast

“…Topoisomerase assays used a derivative of pET28 containing the centromere sequence from Saccharomyces cerevisiae chromosome 3 cloned between EcoRV and XhoI sites. Variations of the ␣-satellite core sequence (16) were used for AUC and obtained by self-priming DNA synthesis (17). The sequence of interest was purified by ion exchange (Mono Q, GE Healthcare) in 50 mM KCl, 50 mM phosphate buffer, pH 6.8, with a gradient to 1 M KCl.…”

Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion

“…Endonuclease-sensitive sites flank the individual dsDNA repeats (45), so that restriction enzyme digestion releases the target dsDNA. Improvements have been reported in terms of sequence flexibility (56) and length of produced target dsDNA (57). It has also been proposed to attain PCR amplification of tandem repeats in a plasmid (53,54).…”

Crystallization of Macromolecules

“…If both the purchasing of purified oligos and in lab sythesis and purification by HPLC are not possible, an alternate protocol for the synthesis and preparation of nucleic acid substrates using basic molecular biology techniques and resources is available (Muecke, 2008). In brief, this technique utilizes self primed amplification of a DNA fragment of interest that is flanked by cohesive end fragments of bacteriophage lambda and restriction sites.…”