Efficient Isolation of Whole Genomic DNA from Cell Cultures and Blood Samples

摘要

It is apparent that many of the functional genetic units in eukaryotic cells encompass large, continuous tracts of DNA. For example, the human factor VIII gene, which encodes the bloodclotting factor deficient in hemophilia A, covers at least 190 kb of DNA. Therefore, in order to characterize these genes at the molecular level, special strategies must be developed to isolate high molecular weight DNA and minimize interruptions in the gene of interest during the DNA isolation procedure. Existing methods for the isolation of high molecular weight genomic DNA, however, suffer from several limitations including poor recovery and shearing of the DNA. Moreover, the use of toxic chemicals in some of these procedures is hazardous and brings unwanted contaminants to the DNA preparations. Alternative nontoxic DNA extraction procedures are, for the most part, time-consuming. In this report we present a safe, rapid and efficient method for the isolation of intact whole genomes from cell cultures and blood specimens. The method uses nucleoid preparations as the source of DNA and exploits their high sedimentation coefficient to separate them away from RNA and proteins by a simple centrifugation step.