Rapid isolation of DNA from complex biological samples using a novel capture reagent--methidium-spermine-sepharose.
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.
Harding
J D
JD
Molecular Biology Research and Development, Bethesda Research Laboratories, Life Technologies Inc., Gaithersburg, MD 20877.
Gebeyehu
G
G
Bebee
R
R
Simms
D
D
Klevan
L
L
D001435:Bacteriophages / Q000032:analysis; D002498:Centrifugation; D004247:DNA / Q000097:blood / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification; D005784:Gene Amplification; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D008863:Microspheres; D012685:Sepharose / Q000031:analogs & derivatives; D013096:Spermine / Q000031:analogs & derivatives; D014556:Urine / Q000032:analysis
D004279:DNA, Viral; C061193:methidiumspermine sepharose; D013096:Spermine; D004247:DNA; D012685:Sepharose