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Circular polymerase extension cloning of complex gene libraries and pathways.

Quan J, Tian J - PLoS ONE (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering & Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America.

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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Related in: MedlinePlus

Gene cloning using CPEC.(A) A schematic diagram of the proposed CPEC mechanism for cloning an individual gene. The vector and the insert share overlapping regions at the ends. After denaturation and annealing (Step 1), the hybridized insert and vector extend using each other as a template until they complete a full circle and reach their own 5′-ends (Step 2). The final completely assembled plasmid has two nicks, one on each strand, at the positions marked by an arrow head. They can be used for transformation (Step 3) with or without further purification. For library cloning, the cycle maybe repeated in order to increase the yield of complete plasmids. (B) CPEC cloning of the lacZα gene. The image shows gel electrophoresis analysis of the CPEC reaction product after 1, 2 and 5 cycles (lanes 1–3). 5 µl of the reaction was separated on a 0.8% agarose gel and visualized after ethidium bromide staining. The assembled full-length plasmid was 2644 bp; the empty vector, 2386 bp. A sequence verified, full-length plasmid purified from a bacteria colony was used as a positive control (lane 4). The upper band (2644 bp) represented the relaxed circular form and the fast-migrating lower band, the closed circular form of the plasmid. The molecular weight marker used in this figure was NEB 1 kb DNA ladder (lane M).
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pone-0006441-g001: Gene cloning using CPEC.(A) A schematic diagram of the proposed CPEC mechanism for cloning an individual gene. The vector and the insert share overlapping regions at the ends. After denaturation and annealing (Step 1), the hybridized insert and vector extend using each other as a template until they complete a full circle and reach their own 5′-ends (Step 2). The final completely assembled plasmid has two nicks, one on each strand, at the positions marked by an arrow head. They can be used for transformation (Step 3) with or without further purification. For library cloning, the cycle maybe repeated in order to increase the yield of complete plasmids. (B) CPEC cloning of the lacZα gene. The image shows gel electrophoresis analysis of the CPEC reaction product after 1, 2 and 5 cycles (lanes 1–3). 5 µl of the reaction was separated on a 0.8% agarose gel and visualized after ethidium bromide staining. The assembled full-length plasmid was 2644 bp; the empty vector, 2386 bp. A sequence verified, full-length plasmid purified from a bacteria colony was used as a positive control (lane 4). The upper band (2644 bp) represented the relaxed circular form and the fast-migrating lower band, the closed circular form of the plasmid. The molecular weight marker used in this figure was NEB 1 kb DNA ladder (lane M).

Mentions: Existing sequence-independent cloning methods require generating complementary single-stranded overhangs between the insert and the vector, a time-consuming and expensive process. We reasoned that it might be possible to eliminate this requirement by using the polymerase extension mechanism to extend double-stranded overlapping insert and vector to form a complete plasmid (Fig. 1A). In this mechanism, the insert and the vector share overlapping sequences on both ends. After denaturation and annealing, the insert and the vector will hybridize and extend using each other as a template to form a complete double-stranded plasmid, leaving only one nick in each strand.


Circular polymerase extension cloning of complex gene libraries and pathways.

Quan J, Tian J - PLoS ONE (2009)

Gene cloning using CPEC.(A) A schematic diagram of the proposed CPEC mechanism for cloning an individual gene. The vector and the insert share overlapping regions at the ends. After denaturation and annealing (Step 1), the hybridized insert and vector extend using each other as a template until they complete a full circle and reach their own 5′-ends (Step 2). The final completely assembled plasmid has two nicks, one on each strand, at the positions marked by an arrow head. They can be used for transformation (Step 3) with or without further purification. For library cloning, the cycle maybe repeated in order to increase the yield of complete plasmids. (B) CPEC cloning of the lacZα gene. The image shows gel electrophoresis analysis of the CPEC reaction product after 1, 2 and 5 cycles (lanes 1–3). 5 µl of the reaction was separated on a 0.8% agarose gel and visualized after ethidium bromide staining. The assembled full-length plasmid was 2644 bp; the empty vector, 2386 bp. A sequence verified, full-length plasmid purified from a bacteria colony was used as a positive control (lane 4). The upper band (2644 bp) represented the relaxed circular form and the fast-migrating lower band, the closed circular form of the plasmid. The molecular weight marker used in this figure was NEB 1 kb DNA ladder (lane M).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713398&req=5

pone-0006441-g001: Gene cloning using CPEC.(A) A schematic diagram of the proposed CPEC mechanism for cloning an individual gene. The vector and the insert share overlapping regions at the ends. After denaturation and annealing (Step 1), the hybridized insert and vector extend using each other as a template until they complete a full circle and reach their own 5′-ends (Step 2). The final completely assembled plasmid has two nicks, one on each strand, at the positions marked by an arrow head. They can be used for transformation (Step 3) with or without further purification. For library cloning, the cycle maybe repeated in order to increase the yield of complete plasmids. (B) CPEC cloning of the lacZα gene. The image shows gel electrophoresis analysis of the CPEC reaction product after 1, 2 and 5 cycles (lanes 1–3). 5 µl of the reaction was separated on a 0.8% agarose gel and visualized after ethidium bromide staining. The assembled full-length plasmid was 2644 bp; the empty vector, 2386 bp. A sequence verified, full-length plasmid purified from a bacteria colony was used as a positive control (lane 4). The upper band (2644 bp) represented the relaxed circular form and the fast-migrating lower band, the closed circular form of the plasmid. The molecular weight marker used in this figure was NEB 1 kb DNA ladder (lane M).
Mentions: Existing sequence-independent cloning methods require generating complementary single-stranded overhangs between the insert and the vector, a time-consuming and expensive process. We reasoned that it might be possible to eliminate this requirement by using the polymerase extension mechanism to extend double-stranded overlapping insert and vector to form a complete plasmid (Fig. 1A). In this mechanism, the insert and the vector share overlapping sequences on both ends. After denaturation and annealing, the insert and the vector will hybridize and extend using each other as a template to form a complete double-stranded plasmid, leaving only one nick in each strand.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering & Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America.

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.

Show MeSH
Related in: MedlinePlus