Method for linking sequences of interest

Details Method for linking sequences of interest Method for linking sequences of interest

C - Chemistry, Metallurgy

12

N

C12N 15/10 (2006.01) C07K 16/12 (2006.01) C12N 15/13 (2006.01)

Patent

CA 2539576

Multiplex overlap-extension RT-PCR provides an efficient method of linking two or more nucleotide sequences encoding for domains or subunits of a heteromeric protein, in a single reaction. Especially, the linkage of variable region encoding sequences from e.g. immunoglobulins, T cell receptors or B cell receptors is eased with the method of the present invention. This allows for a more efficient way of generating libraries of variable region encoding sequences. The capability to perform the multiplex overlap-extension RT-PCR using template derived from an isolated single cell enables the generation of cognate pair libraries in a high-throughput format.

L'invention concerne la possibilité de lier efficacement, par RT-PCR chevauchement-extension multiplex, deux ou plus de deux séquences nucléotidiques codant les domaines ou les sous-unités d'une protéine hétéromère, à travers une seule réaction. En particulier, le procédé de liaison décrit facilite la liaison des séquences de codage à région variable, par exemple à partir des immunoglobulines, des récepteurs de lymphocyte T ou B. Cela permet de produire plus facilement des librairies de telles séquences. La possibilité de mise en oeuvre de RT-PCR chevauchement-extension multiplex au moyen d'un gabarit dérivé d'une cellule isolée simple permet de produire des librairies de paires apparentées sous un fomrat à haut rendement.

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