Preparation of Multimilligram Quantities of Large, Linear DNA Molecules for Structural Studies
| Autor: | Martin A. Samuels, Megan J. Davey, Merlind Muecke, David Jeruzalmi |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Electrophoresis
Agar Gel biology DNA synthesis Base pair Sequence (biology) DNA Chromatography Ion Exchange biology.organism_classification Polymerase Chain Reaction Combinatorial chemistry Molecular biology Bacteriophage chemistry.chemical_compound Endonuclease Sticky and blunt ends chemistry Structural Biology biology.protein Nucleic Acid Amplification Techniques Molecular Biology In vitro recombination |
| Zdroj: | Structure. 16:837-841 |
| ISSN: | 0969-2126 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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