A dot-blot method for quantification of apurinic/apyrimidinic sites in DNA using an avidin plate and liposomes encapsulating a fluorescence dye.
A dot-blot method for quantification of apurinic/apyrimidinic (AP) sites in genomic DNA (calf thymus DNA) is described using an avidin-modified glass slip and biotinylated liposomes containing sulforhodamine B as a fluorescence marker. Aldehyde reactive probe (ARP)-tagged DNA was found to be strongly adsorbed on an avidin slip, even if treated with ethanolamine and biotin, with an efficiency of 51% due to the positive surface charge of avidin, and unbound ARP was easily washed out of the surface with Milli-Q water. In the assay protocol, calf thymus DNA containing AP sites is reacted with ARP in solution and immobilized on an ethanolamine- and biotin-treated avidin slip (EAB-avidin slip), followed by incubation with streptavidin. The AP sites were finally quantified with biotinylated liposomes containing 1.5 mM sulforhodamine B as a fluorescence marker. The mean fluorescence intensity over the surface of the slip was an analytically relevant measure of the amount of AP sites in calf thymus DNA. By using the dot-blot assay, 1-5 AP sites per 10(4) nucleotides in 5 and 100 ng of DNA were quantified. The current dot-blot method has potential for quantification of AP sites in genomic DNA at a level of several nanograms.
Department of Chemistry, College of Humanities Sciences, Nihon University, Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan.
Hiroyuki
H
Yanagisawa
Ayumi
A
Hirano
Masao
M
Sugawara
D000818:Animals; D001360:Avidin / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002417:Cattle; D004247:DNA / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000032:analysis*; D008081:Liposomes / Q000737:chemistry*; D051379:Mice; D011687:Purines / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D019809:Streptavidin
D005456:Fluorescent Dyes; D008081:Liposomes; D011687:Purines; D011743:Pyrimidines; D001360:Avidin; D001710:Biotin; D004247:DNA; D019809:Streptavidin; C427203:calf thymus DNA