Rapid isolation of DNA from complex biological samples using a novel capture reagent--methidium-spermine-sepharose.

AUTOR(ES)

Harding, J D

RESUMO

We have synthesized and analyzed the functional 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.

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