Isolation of Total Cellular DNA from Tissues and Cultured Cells

Abstract

The isolation of high-quality cellular DNA is often a starting point for a variety of molecular-biology techmques These include Southern-blot analysis, PCR amphfication, and genomic-library construction (Fig. 1) It is often necessary to use the DNA from a single preparation for a number of different applrcations Some applications require higher quality DNA than do other applications, such as genomic library construction vs routine polymerase chain reaction (PCR) amplification. Therefore, it is advantageous to employ an isolation procedure that provides high quality DNA Generally, quality is indicated by the absence of contammating RNA, proteins, lipids, and other cellular constituents that may interfere with restriction enzymes, ligases, and thermostable DNA polymerases More importantly, the preparation should be free of contaminating DNA nucleases, which can nick and degrade high-mol-wt DNA The large size of mammahan genomic DNA also requires that the isolation method be gentle enough to minimize mechanical shear stress, which would fragment the large genomic DNA during the course of purification In addition, a good DNA isolation method should be able to accommodate a wide variety of tissues and cell types Many methods have been published that have been optimized for a specific application (1–3), whereas other methods allow the simultaneous isolation of DNA and RNA from the same sample (4,5) Commonly used procedures employ a buffer containing one or several detergents; for example, SDS (6), NP-40, or Triton X-100 (7) These detergents lyse cells and assist in the removal of proteins from the DNA (8).