A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification.

Department of Medical Microbiology, University of Z/irich, 8028 Z/irich, Switzerland A large number of different protocols for the efficient isolation of highly purified DNA from eukaryotic and prokaryotic cells is extant. (1-4) These procedures usually include treatment with proteinase K in the presence of SDS, which efficiently lyses the cells and nuclei and liberates the DNA tightly bound in chromatin. (s) Proteins are then extracted with phenol and chloroform, and the nucleic acids are precipitated with ethanol. This procedure is tedious and time-consuming, and significant amounts of DNA may be lost, especially when working with small specimens (e.g., joint biopsies). Therefore, this approach is not appropriate for diagnostic tests. Direct amplification of digested samples without phenol/chloroform extraction and precipitation is not possible because SDS is inhibitory to Taq polymerase at concentrations as low as 0 . 0 1 % . (6) Alternative simple DNA extraction procedures have been used but have often resulted in incomplete lysis of the cells. These procedures typically have included detergents (e.g., Triton X-100), chaotropes (e.g., guanidium isothiocyanate or sodium iodide), proteases (e.g., proteinase K), substances that lyse erythrocytes and leukocytes (e.g., saponin), or heat denaturation. (7) Often nonionic detergents such as Tween 20 or Laureth 12 in combination with proteinase K are used, followed by heat inactivation of the enzyme prior to PCR amplification. (7-1°) In our laboratory we have aimed at establishing an efficient but simple DNA extraction procedure applicable to various types of clinical specimens, including tissue, sputum, liquid specimens, and bacterial cultures. The procedure would allow direct PCR amplification without purification or precipitation of the DNA. Here, we describe the application of the widely used SDS/proteinase K method made compatible with direct PCR amplification, and including the use of uracil N glycosylase (UNG) to prevent false positives caused by amplicon carryover.( ] 1)