Silica-guanidiniumthiocyanate dna isolation using casein

Details Silica-guanidiniumthiocyanate dna isolation using casein Silica-guanidiniumthiocyanate dna isolation using casein

C - Chemistry, Metallurgy

12

N

C12N 15/10 (2006.01) C07H 1/08 (2006.01) C12P 19/34 (2006.01) C12Q 1/68 (2006.01) C12S 3/20 (2006.01)

Patent

CA 2329515

DNA purified by the guanidiniumthiocyanate-silica procedure (Boom et al, J. Clin. Microbiol. 28:495-503, 1990) from some sources (e.g. cerebrospinal fluid and urine) has appeared to contain inhibitors of DNA processing enzymes (Taq DNA polymerase, restriction enzymes and DNA ligase). These inhibitors were, at least in part, introduced by the purification procedure itself. The inhibition of DNA processing enzymes could be relieved by the inclusion of alpha casein into the guanidiniumthiocyanate (GuSCN) containing lysisbuffer. Alpha casein is copurified with DNA by simultaneous binding to the silica particles and is subsequently eluted together with the DNA in the last step of the purification procedure after which it exerts its beneficial effects for DNA processing enzymes.

Selon l'invention, l'ADN purifié par un procédé à base de guanidiniumthiocyanate-silice (Boom et autres, J. Clin. Microbiol. 28:495-503, 1990), à partir de certaines sources (par exemple le fluide cérébrospinal et l'urine), contient des inhibiteurs des enzymes de catalyse de l'ADN (ADN polymérase taq, enzymes de restriction et ADN ligase). Ces inhibiteurs ont été, au moins en partie, introduits par le procédé de purification lui-même. Il est possible d'éliminer l'inhibition des enzymes de catalyse de l'ADN par l'inclusion d'alpha caséine dans le guanidiniumthiocyanate (GuSCN) contenant le tampon de lyse. On purifie en même temps l'alpha caséine et l'ADN, par leur fixation simultanée sur des particules de silice, puis on élue ensemble l'alpha caséine avec l'ADN dans la dernière étape du procédé de purification, après quoi cette alpha caséine peut exercer ses effets bénéfiques sur les enzymes de catalyse de l'ADN.

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