Rapid generation of long synthetic centromeric tandem...

C - Chemistry – Metallurgy – 12 – Q

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C12Q 1/68 (2006.01) C12N 15/85 (2006.01)

Patent

CA 2616085

Methods are described for construction of long synthetic arrays of DNA repeats, such as alphoid repeats or other repeat sequences. The methods include concatamerization of DNA into short repeats (for instance using rolling circle amplification or directional in vitro ligation), followed by assembling the short repeats into long arrays by homologous recombination during transformation into microbe cells. These methods can be described generally as Recombinational Amplification of Repeats (RAR). The long arrays are engineered centromere-like regions that allow one to construct mammalian artificial chromosomes with a predefined centromeric region structure. Artificial chromosomes, including human artificial chromosomes with a regulated centromere, and methods of their use are also provided

La présente invention concerne des procédés de construction de réseaux synthétiques longs d'unités de répétition d'ADN, notamment des unités de répétition d'alphoïdes ou d'autres séquences d'unités de répétition. Le procédé comporte une concatamérisation de l'ADN en courtes unités de répétition, par exemple par amplification circulaire roulante ou ligation directionnelle in vitro, suivie d'un assemblage de ces courtes unités de répétition en réseaux longs par recombinaison d'homologues pendant la transformation en cellules microbiennes. Ces procédés constituent de l'amplification recombinatoire d'unités de répétition ou "RAR" (Recombinational Amplification of Repeats). Ces réseaux longs, qui sont des régions ressemblant à des centromères obtenus par génie génétique, permettent de construire, en respectant une structure prédéfinie de région centromère, des chromosomes mammaliens artificiels. L'invention concerne également des chromosomes artificiels, y-compris des chromosomes artificiels humains à centromère régulé, ainsi que les procédés d'utilisation correspondants.

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