Nucleic Acids Research, 1989, Vol. 17, No. 17 6947-6958
© 1989
CHEMISTRY |
Rapid isolation of DNA from complex biological samples ustng a novel capture reagent—methidium spermine-sepharose
Molecular Biology Research and Development, Bethesda Research Laboratories, Life Technologies Inc. 8717 Grovemont Circle, Gaithersburg, MD 20877, USA
*To whom correspondence should be addressed
Received May 17, 1989. Accepted August 4, 1989.
We have synthesized and analyzed the f unctional properties of a novel DNA capture reagent containing a methidium moiety attached to a sepharose bead by a spermine linker. DNA present in a biological fluid or other complex sample binds to the reagent. The DNA-capture reagent complex is then separated from the sample by centrifugation and the DNA is released from the reagent by brief incubation in 0.1 to 0.5 N NaOH or KOH. Capture of DNA from complex samples is independent of the salt concentration of the sample, and occurs in the presence of high concentrations of EDTA, proteinase K and detergents. Many samples can be processed simultaneously. The following specific applications, in which denatured DNA is quantitated or characterized, are demonstrated: 1). Isolation of hepatitis B virus DNA from serum and quantitation by dot-blot hybridization, 2). Isolation and quantitation of DNA from urine, 3). Isolation of human genomic DNA from one microliter of blood or 100 HeLa cells followed by amplification of a specific gene sequence using the Polymerase Chain Reaction, 4). Isolation of single stranded phage M13 sequencing templates from bacterial cultures. These investigations suggest that a capture reagent containing an intercalating moiety bound to a solid support may be useful for many applications in molecular biology and molecular diagnostics.