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      Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways

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      Author(s): , *
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      Journal: PLoS ONE
      Publisher: Public Library of Science

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          Abstract

          High-throughput genomics and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries and synthetic pathways. Here, we describe the development of a novel and extremely simple cloning method, circular polymerase extension cloning (CPEC). This method uses a single polymerase to assemble and clone multiple inserts with any vector in a one-step reaction in vitro. No restriction digestion, ligation, or single-stranded homologous recombination is required. In this study, we elucidate the CPEC reaction mechanism and demonstrate its usage in demanding synthetic biology applications such as one-step assembly and cloning of complex combinatorial libraries and multi-component pathways.

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          Most cited references22

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          Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.

          Mamie Z Li, Stephen J. Elledge (2007)
          We describe a new cloning method, sequence and ligation-independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. SLIC inserts can also be prepared by incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and reproducible assembly of recombinant DNA with as many as 5 and 10 fragments simultaneously. SLIC circumvents the sequence requirements of traditional methods and functions much more efficiently at very low DNA concentrations when combined with RecA to catalyze homologous recombination. This flexibility allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.
          Article 0 comments Cited 338 times – based on 0 reviews     Review now
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            DNA cloning using in vitro site-specific recombination.

            J. L. Hartley, G Temple, M. A. Brasch (2000)
            As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.
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              Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides.

              Cynthia Pfannkoch, J. Craig Venter, Colin A. Hutchison … (2003)
              We have improved upon the methodology and dramatically shortened the time required for accurate assembly of 5- to 6-kb segments of DNA from synthetic oligonucleotides. As a test of this methodology, we have established conditions for the rapid (14-day) assembly of the complete infectious genome of bacteriophage X174 (5386 bp) from a single pool of chemically synthesized oligonucleotides. The procedure involves three key steps: (i). gel purification of pooled oligonucleotides to reduce contamination with molecules of incorrect chain length, (ii). ligation of the oligonucleotides under stringent annealing conditions (55 degrees C) to select against annealing of molecules with incorrect sequences, and (iii). assembly of ligation products into full-length genomes by polymerase cycling assembly, a nonexponential reaction in which each terminal oligonucleotide can be extended only once to produce a full-length molecule. We observed a discrete band of full-length assemblies upon gel analysis of the polymerase cycling assembly product, without any PCR amplification. PCR amplification was then used to obtain larger amounts of pure full-length genomes for circularization and infectivity measurements. The synthetic DNA had a lower infectivity than natural DNA, indicating approximately one lethal error per 500 bp. However, fully infectious X174 virions were recovered after electroporation into Escherichia coli. Sequence analysis of several infectious isolates verified the accuracy of these synthetic genomes. One such isolate had exactly the intended sequence. We propose to assemble larger genomes by joining separately assembled 5- to 6-kb segments; approximately 60 such segments would be required for a minimal cellular genome.
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                Author and article information

                Contributors
                : Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2009
                Publication date (Electronic): 30 July 2009
                Volume: 4
                Issue: 7
                Electronic Location Identifier: e6441
                Affiliations
                [1]Department of Biomedical Engineering & Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America
                Instituto Butantan, Brazil
                Author notes

                Conceived and designed the experiments: JT. Performed the experiments: JQ JT. Analyzed the data: JQ JT. Wrote the paper: JT.

                Article
                Publisher ID: 09-PONE-RA-10278R1
                DOI: 10.1371/journal.pone.0006441
                PMC ID: 2713398
                PubMed ID: 19649325
                SO-VID: bede1891-7589-4b73-a43d-05d7a91d91f8
                Copyright © Quan, Tian. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                Date received : 10 May 2009
                Date accepted : 29 June 2009
                Page count
                Pages: 6
                Categories
                Subject: Research Article
                Subject: Biotechnology
                Subject: Genetics and Genomics
                Subject: Molecular Biology
                Subject: Biotechnology/Applied Microbiology
                Subject: Biotechnology/Bioengineering
                Subject: Biotechnology/Plant Biotechnology
                Subject: Cell Biology/Gene Expression
                Subject: Genetics and Genomics/Cancer Genetics
                Subject: Genetics and Genomics/Functional Genomics
                Subject: Genetics and Genomics/Gene Expression
                Subject: Genetics and Genomics/Genomics

                ScienceOpen disciplines: Uncategorized
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                ScienceOpen disciplines: Uncategorized

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