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Nucleic Acids Research, 1992, Vol. 20, No. 19 5067-5072
© 1992

MOLECULAR BIOLOGY

Identification of a DNA supercoiling activity in Saccharomyces cerevisiae

Hyeon-Sook Koo+, Kawai Lau, Hai-Young Wu§ and Leroy F. Liu*

Department of Biological Chemistry, Johns Hopkins Medical School 725 N.Wolfe St., Baltimore, MD 21205, USA

* To whom correspondence should be addressed

Received July 9, 1992. Revised August 28, 1992. Accepted August 28, 1992.

A sensitive and simple method for the quantitatlon of human DNA is described. This method Is based on probe hybridization to a human alpha satellite locus, D17Z1. The biotlnylated probe is hybridized to sample DNA immobilized on nylon membrane. The subsequent binding of streptavldln-horseradish peroxidase to the bound probe allows for chemlluminescent detection using a luminol-based reagent and X-ray film. Less than 150 pg of human DNA can easily be detected with a 15 minute exposure. The entire procedure can be performed in 1.5 hours. Microgram quantities of non-human DNA have been tested and the results indicate very high specificity for human DNA. The data on film can be scanned into a computer and a commercially available program can be used to create a standard curve where DNA quantity is plotted against the mean density of each slot blot signal. The methods described can also be applied to the very sensitive determination of quantity and quality (size) of DNA on Southern blots. The high sensitivity of this quantltation method requires the consumption of only a fraction of sample for analysis. Determination of DNA quantity is necessary for RFLP and many PCR-based tests where optimal results are obtained only with a relatively narrow range of DNA quantities. The specificity of this quantitation method for human DNA will be useful for the analysis of samples that may also contain bacterial or other non-human DNA, for example forensic evidence samples, ancient DNA samples, or clinical samples.

+Department of Biochemistry, Yonsei University, College of Science, Seodflemungu Sinchondong 134, Seoul 120-749, Korea

§Department of Pharmacology, Wayne State University, School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201, USA


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