Preparation of multimilligram quantities of large, linear DNA molecules for structural studies.
Muecke M, Samuels M, Davey M, Jeruzalmi D
Structure (2008)
Category: DNA structure, methods ¤ Added: Jul 09, 2008 ¤ Rating: ◊◊
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 frombacteriophage lambda as well as endonuclease recognition sites. Amplification produces long polymers of DNA, tens of kilobases in length, which harbormany copies of the sequence of interest. Endonuclease digestion of these polymers, followed by chromatographic purification, yields high-quality preparations of theDNAmolecule of interest.Reliance on the cohesive end sequences to initiate self-primed amplification effectively enables the synthesis of DNA molecules of interestwithminimal restriction on length and sequence.