Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.ip68

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Strategies for Cloning PCR Products

Peter D’Arpa

Adapted from PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). CSHL Press, Cold Spring Harbor, NY, USA, 2003.


INTRODUCTION

Cloning polymerase chain reaction (PCR)-amplified fragments into plasmids offers several advantages. Bacteria containing plasmids can be frozen, providing a ready supply of amplified material. Because of the variety of available plasmids with different promoters and selectable markers, cloning is also useful when mutations are to be introduced into the fragment before expression, or when sequence tags encoded in the vector are to be added in-frame. The ease with which nucleotide sequences can be added to the ends of PCR products has led to the development of a variety of cloning strategies. Because such cloning is typically the first step for generating a reagent that will be used to achieve a specific experimental goal, the efficiency of the cloning procedure is an important consideration: Cloning strategies should be simple in design and execution, requiring a minimum of enzymatic steps. Toward this goal, many companies market and continue to develop reagent kits that improve the ease and rapidity of cloning PCR products. This article focuses on some common and efficient cloning strategies, such as those that use DNA ligase or vaccinia virus topoisomerase I (TOPO), as well as techniques for in vitro and in vivo recombination of PCR products and vectors having homologous duplex ends. Also covered is the production of linear PCR products with defined 5' and 3' functional elements, which enable direct mammalian cell expression or in vitro transcription/translation. We present an overview of these strategies, their molecular basis, and their advantages and disadvantages for specific applications.


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