Preparation of multimilligram quantities of large, linear DNA molecules for structural studies.
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.
Muecke
Merlind
M
Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.
Samuels
Martin
M
Davey
Megan
M
Jeruzalmi
David
D
D002852:Chromatography, Ion Exchange; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction
D004247:DNA