Research Paper | Published:

A Universal Method for the Direct Cloning of PCR Amplified Nucleic Acid

Bio/Technologyvolume 9pages657663 (1991) | Download Citation

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Abstract

We have devised a simple, universal cloning strategy that permits the direct liga-tion of PCR amplified nucleic acid to a compatible vector preparation. The method does not require that special restriction sites or additional sequences be appended onto the amplification primers, nor the use of restriction endonucleases, modifying enzymes, or any purification procedures. This approach takes advantage of the single 3′ deoxyadenylate extension that Thermus aquations, Thermus flavus, and Thermococcus litoralis DNA polymerases add to the termini of amplified nucleic acid. A new type of plasmid was constructed that allows the preparation of ends containing a single complementary 3′ deoxythymidylate extension. Cloning PCR products by this method is approximately 50 times more efficient than blunt-ended ligation reactions. This direct PCR ligation technique has been engineered in-phase with a truncated lacZ gene to facilitate the discrimination of recombinants from non-recombinants. We have applied this method to directly clone amplified RNA and DNA from as little as 1 μl of a PCR reaction, without prior modification or purification steps.

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Author notes

  1. Lloyd M. Smith: Corresponding author.

Affiliations

  1. Department of Chemistry, University of Wisconsin, Madison, WI, 53706

    • David A. Mead
    • , N. Kristy Pey
    •  & Lloyd M. Smith
  2. Invitrogen Corporation, 11588 Sorrento Valley Rd., Suite 20, San Diego, CA, 92121

    • Corinna Herrnstadt
    •  & Robert A. Marcil

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DOI

https://doi.org/10.1038/nbt0791-657

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