Isolation of High-Molecular-Weight DNA from Animal Cells

Mammalian chromosomes are of the order of 12–60 times the size of that of Escherichia coli (4 � 10³ kilobase pairs [kbp]) (1 ). The choice of method used when purifying DNA from mammalian cells may be dictated by the use to which the product will be put as it will influence the average size of the material purified. For example, methods incorporating many aggressive manipulations will tend to shear the DNA into molecules of relatively low-mol-wt (< 50 kbp). This may be suitable for polymerase chain reaction (PCR) (2 ,3 ) analysis and in some cases Southern blotting (4 ) but will be unsuitable for other more demanding purposes, e.g., genomic library constructions. When performed with care, methods involving minimal manipulations will yield DNA in excess of 200 kbp, suitable for most purposes. In general, it is prudent to utilize such methods for all preparations of DNA from mammalian cells. The first three methods described below are derived from that of Blin and Stafford (5 ) and should yield high-mol-wt (HMW) DNA from solid tissues, blood, or cells in culture suitable for most purposes including cloning, PCR/RFLP analysis, and Southern blotting. The final method described is that of Lahiri and Nurnberger (6 ) and is a rapid approach that eliminates the use of solvents and enzymes, making it easier to process large numbers of samples. The material produced by this method should be approx 50 kbp and is suitable for PCR and RFLP analysis.