Partially double-stranded nucleic acids, methods of making,...

Details Partially double-stranded nucleic acids, methods of making,... Partially double-stranded nucleic acids, methods of making,...

C - Chemistry, Metallurgy

12

Q

C12Q 1/68 (2006.01)

Patent

CA 2427319

A process is disclosed for generating at least one partially double-stranded target nucleic acid, which contains at least one single-stranded region at a tenninal end. The process comprises the steps of (a) providing at least one primer, P1, containing at least one labile nucleotide; (b) combining at least one target nucleic acid sequence with P1 to generate a double-stranded polynucleotide containing at least one labile nucleotide; (c) exposing the double-stranded polynucleotide to conditions that promote single-strand cleavage of the polynucleotide at the site of the at least one labile nucleotide of primer P1; and (d) exposing the cleaved polynucleotide to conditions that promote the dissociation of the cleaved portions of primer PI from a terminal end. The labile nucleotide may be dUTP, wherein the single- stranded cleavage of the polynucleotide at the site of the labile nucleotide occurs by treatment with uracil N-glycosylase.

L'invention concerne un procédé permettant de générer au moins un acide nucléique cible partiellement bicaténaire comportant au moins une région monocaténaire à une extrémité terminale. Le procédé comprend les étapes consistant à (a) fournir au moins une amorce, P1, comprenant au moins un nucléotide labile ; (b) combiner au moins une séquence d'acide nucléique cible avec P1 pour générer un polynucléotide bicaténaire comportant au moins un nucléotide labile ; (c) exposer le polynucléotide bicaténaire à des conditions favorisant le clivage monocaténaire du polynucléotide au niveau du site du ou des nucéotide(s) labile(s) de l'amorce P1 ; et (d) exposer le polynucléotide clivé à des conditions favorisant la dissociation des parties clivées de l'amorce P1 d'une extrémité terminale. Le nucléotide labile peut être du type dUPT. Le clivage monocaténaire du polynucléotide au niveau du site du nucléotide labile s'effectue par traitement par de l'uracile N-glycosylase.

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