Large DNA cloning system based on yeast artificial chromosomes
AssigneeWashington University
InventorsMaynard V. Olson, David T. Burke
Life of this patent
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Abstract
A large DNA cloning system is disclosed which is based on yeast artificial chromosomes. Cloning vectors are disclosed which allow the cloning of large segments of greater than 50 kb of exogenous DNA. The cloning vector comprises DNA sequences of an autonomous replication sequence, a centromere, a selectable yeast marker, two sequences that seed telomere function in vivo, and a cloning site within an interruptible yeast gene for insertion of the exogenous DNA segments.
Claims21
A Saccharomyces yeast artificial chromosome circular vector comprising the DNA sequences of an autonomous replication sequence, a centromere, a selectable yeast marker, two sequences that seed telomere formation in vivo, and a cloning site for insertion of large segments of greater than 50 kb of exogenous DNA within a yeast gene whose interruption is phenotypically visible.
A Saccharomyces yeast artificial chromosome circular vector comprising the DNA sequences of an autonomous replication sequence, a centromere, selectable yeast markers on both sides of the centromere, two sequences that seed telomere formation in vivo separated by a throw-away sequence, and a cloning site for insertion of large segments of greater than 50 kb of exogenous DNA within a yeast gene whose interruption is phenotypically visible.
Plasmid pYAC2.
Plasmid pYAC4.
The method of producing a yeast artificial chromosome comprising cleaving plasmid pYAC2 with BamH I and Sma I restriction endonucleases to form a left chromosome arm including the centromere and the autonomous replication sequence, a right chromosomal arm, and a throw-away sequence, treating said arms with alkaline phosphatase to prevent religation and then ligating said arms onto a large exogenous DNA inset molecule of greater than 50 kb having Sma I compatible ends.
The method of producing a yeast artificial chromosome comprising cleaving plasmid pYAC4 with BamH I and EcoR I restriction endonucleases to form a left chromosome arm including the centromere and the autonomous replication sequence, a right chromosomal arm, and a throw-away sequence, treating said arms with alkaline phosphatase to prevent religation and the ligating said arms onto a large exogenous DNA insert molecule of greater than 50 kb having SnaB I compatible ends.
Dependencies are inferred from the claim text.