Direct cloning of DNA fragments
AssigneeNovagen, Inc.
InventorsRobert C. Mierendorf, Robert E. Novy, Kristin M. Kolb, David O'Reilly
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Abstract
A vector for the direct cloning of the products of PCR protocol incorporates single nucleotide overhangs at one or both ends of a linearized DNA segment. The single nucleotide overhangs are uracil or inosine residues, as desired, to facilitate cloning of the desired PCR products.
Claims12
A DNA vector comprising a linearized double stranded DNA molecule with a single unpaired nucleotide overhang at at least one 3' end thereof, the single overhanging nucleotide being selected from the group consisting of a uracil residue and an inosine residue.
A kit for the direct cloning of PCR products comprising
- an aliquot of a DNA vector comprising a linearized double stranded DNA molecule with a single unpaired nucleotide overhang at each 3' end thereof, the single overhanging nucleotide being selected from the group consisting of a uracil residue and an inosine residue;
- and
- an aliquot of a DNA ligase which catalyzes ligation between the DNA vector and PCR products.
A kit for the direct cloning of PCR products comprising
- an aliquot of a DNA vector comprising a linearized double stranded DNA molecule with a single unpaired nucleotide overhang at at least one 3' end thereof, the single overhanging nucleotide being selected from the group consisting of a uracil residue and an inosine residue;
- and
- an aliquot of competent cells which hosts the vector for replication once a PCR product has been ligated into the vector.
A method of direct cloning of PCR products for a target DNA sequence comprising the steps of
- performing a polymerase chain reaction procedure to amplify copies of a target DNA sequence producing a plurality of PCR products with single nucleotide overhangs at each end thereof;
- forming a ligation mixture including the PCR products and a linear DNA vector having a single unpaired nucleotide overhang at at least one 3' end thereof, the single overhanging nucleotide being selected from the group consisting of a uracil residue and an inosine residue, the ligation mixture being under conditions favoring ligation of the PCR products not the DNA vector;
- and
- transforming the ligated vector into a competent host.
Dependencies are inferred from the claim text.