Enzymatic synthesis of ssdna

Details Enzymatic synthesis of ssdna Enzymatic synthesis of ssdna

C - Chemistry, Metallurgy

12

N

C12N 15/10 (2006.01) A61K 31/711 (2006.01) A61K 48/00 (2006.01) C12N 5/10 (2006.01) C12N 15/11 (2006.01) C12N 15/85 (2006.01)

Patent

CA 2346155

Methods and compositions for producing single-stranded cDNA (ss-cDNA) with a vector-based system in eukaryotic cells. The vector contains all necessary signaling instructions and enzymatic functions to allow the host cell to produce the ssDNA encoding a desired nucleic acid sequence (a "sequence of interest"). Described are the components included in the vector for synthesizing ssDNA in vivo. They include (1) a reverse transcriptase gene, (2) a genetic element that supplies the template for the desired ssDNA sequence of interest, and (3) a second genetic element located proximal to the genetic element encoding the sequence of interest that supplies the primer site for reverse transcription by the reverse transcriptase molecule. The vector also contains appropriate promoter(s)/enhancer(s). Also described herein is a method to construct a vector including these components.

L'invention concerne des méthodes et compositions de production d'ADN complémentaire simple brin grâce à un système vectoriel dans des cellules eucaryotes. Le vecteur contient toutes les instructions de signalisation et le fonctions enzymatiques nécessaires pour permettre à la cellule hôte de produire l'ADN simple brin codant pour une séquence d'acide nucléique voulue (une <= séquence intéressante >=). L'invention concerne également les composants compris dans le vecteur et destinés à synthétiser l'ADN simple brin in vivo. Ces composants comprennent: (1) un gène de transcriptase inverse, (2) un élément génétique fournissant la matrice de la séquence d'ADN simple brin intéressante et (3) un deuxième élément génétique situé à proximité de l'élément génétique codant pour la séquence intéressante qui fournit le site d'amorçage de la transcription inverse par la molécule de transcriptase inverse. Le vecteur contient également des promoteurs/séquences activatrices appropriées. L'invention concerne enfin une méthode de mise au point d'un vecteur comprenant ces composants.

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