Analytical Biochemistry 471 (2015) 80–82
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Universal CG cloning of polymerase chain reaction products Julian Stevenson a,b, Andrew J. Brown a,⇑ a b
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia Department of Nutrition and Toxicology, University of California, Berkeley, CA 94720, USA
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 26 October 2014 Accepted 28 October 2014 Available online 4 November 2014 Keywords: PCR Cloning Plasmid vectors
a b s t r a c t Single-insert cloning of DNA fragments without restriction enzymes has traditionally been achieved using TA cloning, with annealing of a polymerase chain reaction (PCR) fragment containing a single overhanging 30 A to a plasmid vector containing a 30 T. In this article, we show that the analogous ‘‘CG cloning’’ is faster and far more efficient, using AhdI to generate a C-vector. For an afternoon ligation, CG cloning achieved double the cloning efficiency and more than 4-fold the number of transformants compared with TA cloning. However, blunt-end ligation was markedly more efficient than both. CG cloning could prove to be extremely useful for single-copy high-throughput cloning. Ó 2014 Elsevier Inc. All rights reserved.
With improvements in high-throughput sequencing technology, there is an increasing need to efficiently clone DNA. Isolation of fragments is traditionally achieved using polymerase chain reaction (PCR)1 and numerous manipulations, including the addition of linkers, restriction endonuclease digestion, and ligation into plasmid vectors. Blunt-end ligation requires no additional sequences but requires the use of a proofreading polymerase or polishing of the ends before ligation and can also suffer from the problem of cloning multiple copies of an insert. Concatemers and chimeras can significantly reduce the quality of random DNA libraries used in some high-throughput sequencing pipelines. To address this limitation, TA cloning was developed [1], where fragments with single-basepair overhangs are prepared for ligation. This is made possible by the ability of Taq DNA polymerase to add a single non-templatedependent 30 deoxyadenosine (A) to PCR products, which can then anneal to a plasmid vector backbone fragment containing a single overhanging T. Taq polymerase can also add other nucleotides [2–4], which raises the possibility of preparing a G-tailed product for use with a C-vector. This may be useful for cloning in that it would allow the formation of a stronger C:G base-paired junction containing an additional hydrogen bond to the T:A base pair (3 vs. 2). This may consequently improve annealing of single-nucleotide overhangs and, thus, increase cloning efficiency. To test this, we prepared a vector that could be treated to yield a single C, T, or no overhang and cloned a reporter resistance cassette to gauge cloning efficiency, for example, through ligation of a C-vector with a G-tailed insert
⇑ Corresponding author. 1
E-mail address: [email protected] (A.J. Brown). Abbreviation used: PCR, polymerase chain reaction.
http://dx.doi.org/10.1016/j.ab.2014.10.018 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
(Fig. 1). Tandem AhdI sites [5] separated by an 8-bp random linker sequence were introduced into pcDNA4 Myc His B (Life Technologies) using primers 50 -GACTGTCGGTCGCGAGTATGACCGACAGTCCA GCACAGTGGCGGCCGCTCGAGT-30 and 50 -GACTGTCGGTCATACTCGC GACCGACAGTCGATATCTGCAGAATTCCACCAC-30 or 50 - GACTGCC GGTCGCGAGTATGACCGGCAGTCCAGCACAGTGGCGGCCGCTCGAGT-30 and 50 -GACTGCCGGTCATACTCGCGACCGGCAGTCGATATCTGCAGAATTCCACCAC-30 (recognition sequences are underlined, and the resulting overhanging base is in boldface and italicized) with polymerase incomplete primer extension site-directed mutagenesis [6,7] with Phusion High Fidelity Polymerase (all enzymes from New England Biolabs) using a lower primer concentration (0.2 lM instead of 0.5 lM) because primer dimer formation due to the presence of long complementary 50 overhangs led to no amplification. AhdI was selected because it cuts any sequence between its two recognition sequences and can be used for prolonged digestion without star activity. Silent site-directed mutagenesis [8,9] of the ampicillin resistance gene was also performed to remove an existing AhdI restriction site using primers T7 F and 50 -CGTAGTTATCTACACAACGGGGAGCCAGGCAACTATGG-30 . Vectors (3 lg) were digested with 10 U of AhdI (for single-nucleotide overhangs) or EcoRV HF (for blunt ends) for 16 h in CutSmart Buffer at 37 °C, incubated for a further 1 h with 10 U of Antarctic Phosphatase, and purified using a HiYield Gel/PCR DNA Mini Kit (Real Biotech). A 1.4-kb kanamycin resistance cassette from pUC18/Kan was prepared by PCR using phosphorylated primers pUC18-Kan F and pUC18-Kan R and Phusion High Fidelity Polymerase [7]. Purification and treatment of blunt product with dGTP is highly recommended for CG cloning because Taq strongly prefers to add a 30 A [3]. This is unlikely to require additional steps compared with TA cloning because proofreading
Notes & Tips / Anal. Biochem. 471 (2015) 80–82
81
Fig.1. Principles of CG cloning of a PCR product. A vector engineered to include two AhdI cleavage sites within the multiple cloning site (MCS) is digested to generate a linear fragment containing a single overhanging 30 C. Phosphatase treatment helps to eliminate empty vector background by preventing ligation of any singly cut vector or DNA, which loses the overhang over time. Blunt PCR product is either phosphorylated or prepared using phosphorylated primers, purified to remove dNTPs, and then treated with Taq DNA polymerase and dGTP to add single overhanging 30 Gs to the insert. Insert and vector are mixed and anneal via the single-base pairs. A nicked recombinant plasmid is then formed by T4 DNA ligase, which is transformed into Escherichia coli, which in this example will now be resistant to kanamycin due to the incorporation of the insert containing the KanR gene.
Fig.2. Direct comparison of TA, CG, and blunt-end cloning. See text for details. The average cloning efficiency is marked for clarity. Error bars are standard errors for the number of colonies for three independent experiments.
polymerases are routinely used for the initial amplification step to avoid errors. Gel-purified PCR product (500 ng) was treated with 200 lM dATP or dGTP using 5 U of Taq DNA polymerase for 30 min at 72 °C (or untreated for use in blunt-end ligation). Tailed product was column purified again to control for a possible inhibitory effect of dATP on ligation. Ligation used 50 ng each of insert and vector and 400 U of T4 DNA ligase at 15 °C for 30 min, 3 h, or 16 h. Next, 25-ll aliquots of XL10 Gold cells (Agilent) were transformed with 2.5 ll of ligation mix according to the manufacturer’s instructions. Cells were plated onto selective LB/agar. Cloning efficiency—the percentage of successful recombinants—was determined by first plating cells onto LB/agar containing ampicillin and then patching 26 clones onto kanamycin selective plates to confirm the presence of the KanR cassette. We also tried alternative strategies to prepare C-vector as previously used to create T-vectors [2]. Unacceptably low cloning efficiencies were obtained after attempts to add single overhanging Ts and Cs to blunt linearized vector using Taq polymerase or ddTTP/ ddCTP with terminal transferase, likely due to the poor tailing efficiency of Taq or the inability to phosphatase treat the vector, respectively. We directly compared TA, CG, and blunt-end cloning using the restriction-enzyme-treated vectors (Fig. 2). For shorter ligation times (30 min and 3 h), TA cloning performed poorly, yielding very low efficiencies (15 and 38%, respectively), although overnight ligation achieved useful efficiencies (74%). On the contrary, CG cloning achieved high efficiencies for all ligation times and approximately 4-fold more transformants than TA cloning. Thus, CG cloning can allow ligation and transformation on the same day and
with greater robustness than TA cloning. Blunt-end cloning performed the best, yielding close to 100% efficiency and more than 20-fold more colonies than TA cloning, reflecting the difficulty of ligating annealed single-base-pair overhangs. CG cloning is faster and more efficient than TA cloning, but for single genes blunt-end cloning using a proofreading polymerase is likely to be sufficient and most convenient because it does not require a tailing step and does not suffer from the high error rates observed using Taq DNA polymerase. However, none of these methods achieves directional cloning. Alternatively, ligation-independent cloning methods are simpler and more robust than cloning with restriction enzymes and ligation and achieve very high efficiencies with limited manipulations for a wide range of insert sizes [7], but they require the addition of long 50 overhangs to PCR primers, increasing the cost of cloning projects. However, for higher throughput cloning of short sequences for library construction, which do not tolerate the presence of concatemers, CG cloning is most suitable. During the preparation of this article, we became aware of commercially available kits developed by Lucigen using this principle [10], which the manufacturer refers to as ‘‘GC cloning,’’ with their own pSMART GC and pGC Blue vectors—and with additional steps to help eliminate empty vector background. As shown here in controlled experiments, CG cloning is indeed superior to TA cloning, and any C-vector can easily be prepared in-house. Acknowledgments The Brown Laboratory is supported by grants from the National Health and Medical Research Council (1008081) and the National
82
Notes & Tips / Anal. Biochem. 471 (2015) 80–82
Heart Foundation of Australia (G11S5757). The authors thank members of the Brown Laboratory for critically reviewing the manuscript. References [1] D.A. Mead, N.K. Pey, C. Herrnstadt, R.A. Marcil, L.M. Smith, A universal method for the direct cloning of PCR amplified nucleic acid, Biotechnology (N. Y.) 9 (1991) 657–663. [2] M.Y. Zhou, C.E. Gomez-Sanchez, Universal TA cloning, Curr. Issues Mol. Biol. 2 (2000) 1–7. [3] J.M. Clark, Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases, Nucleic Acids Res. 16 (1988) 9677–9686. [4] G. Hu, DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 30 end of a DNA fragment, DNA Cell Biol. 12 (1993) 763–770. [5] J.U. Jeung, S.K. Cho, K.S. Shim, S.H. Ok, D.S. Lim, J.S. Shin, Construction of two pGEM-7Zf(+) phagemid T-tail vectors using AhdI restriction endonuclease sites for direct cloning of PCR products, Plasmid 48 (2002) 160–163.
[6] H.E. Klock, E.J. Koesema, M.W. Knuth, S.A. Lesley, Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts, Proteins 71 (2008) 982–994. [7] J. Stevenson, J.R. Krycer, L. Phan, A.J. Brown, A practical comparison of ligationindependent cloning techniques, PLoS ONE 8 (2013) e83888. [8] J. Sanchis, L. Fernandez, J.D. Carballeira, J. Drone, Y. Gumulya, H. Hobenreich, D. Kahakeaw, S. Kille, R. Lohmer, J.J. Peyralans, J. Podtetenieff, S. Prasad, P. Soni, A. Taglieber, S. Wu, F.E. Zilly, M.T. Reetz, Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates, Appl. Microbiol. Biotechnol. 81 (2008) 387–397. [9] W.C. Tseng, J.W. Lin, T.Y. Wei, T.Y. Fang, A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method, Anal. Biochem. 375 (2008) 376– 378. [10] D. A. Mead, R. Godiska, T. W. Schoenfeld, S. Hermanson, Plasmid comprising single stranded overhangs for use in replication of preferential gene sequences, patent US 7723103 BW, 2010.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Universal CG cloning of polymerase chain reaction products Julian Stevenson a,b, Andrew J. Brown a,⇑ a b
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia Department of Nutrition and Toxicology, University of California, Berkeley, CA 94720, USA
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 26 October 2014 Accepted 28 October 2014 Available online 4 November 2014 Keywords: PCR Cloning Plasmid vectors
a b s t r a c t Single-insert cloning of DNA fragments without restriction enzymes has traditionally been achieved using TA cloning, with annealing of a polymerase chain reaction (PCR) fragment containing a single overhanging 30 A to a plasmid vector containing a 30 T. In this article, we show that the analogous ‘‘CG cloning’’ is faster and far more efficient, using AhdI to generate a C-vector. For an afternoon ligation, CG cloning achieved double the cloning efficiency and more than 4-fold the number of transformants compared with TA cloning. However, blunt-end ligation was markedly more efficient than both. CG cloning could prove to be extremely useful for single-copy high-throughput cloning. Ó 2014 Elsevier Inc. All rights reserved.
With improvements in high-throughput sequencing technology, there is an increasing need to efficiently clone DNA. Isolation of fragments is traditionally achieved using polymerase chain reaction (PCR)1 and numerous manipulations, including the addition of linkers, restriction endonuclease digestion, and ligation into plasmid vectors. Blunt-end ligation requires no additional sequences but requires the use of a proofreading polymerase or polishing of the ends before ligation and can also suffer from the problem of cloning multiple copies of an insert. Concatemers and chimeras can significantly reduce the quality of random DNA libraries used in some high-throughput sequencing pipelines. To address this limitation, TA cloning was developed [1], where fragments with single-basepair overhangs are prepared for ligation. This is made possible by the ability of Taq DNA polymerase to add a single non-templatedependent 30 deoxyadenosine (A) to PCR products, which can then anneal to a plasmid vector backbone fragment containing a single overhanging T. Taq polymerase can also add other nucleotides [2–4], which raises the possibility of preparing a G-tailed product for use with a C-vector. This may be useful for cloning in that it would allow the formation of a stronger C:G base-paired junction containing an additional hydrogen bond to the T:A base pair (3 vs. 2). This may consequently improve annealing of single-nucleotide overhangs and, thus, increase cloning efficiency. To test this, we prepared a vector that could be treated to yield a single C, T, or no overhang and cloned a reporter resistance cassette to gauge cloning efficiency, for example, through ligation of a C-vector with a G-tailed insert
⇑ Corresponding author. 1
E-mail address: [email protected] (A.J. Brown). Abbreviation used: PCR, polymerase chain reaction.
http://dx.doi.org/10.1016/j.ab.2014.10.018 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
(Fig. 1). Tandem AhdI sites [5] separated by an 8-bp random linker sequence were introduced into pcDNA4 Myc His B (Life Technologies) using primers 50 -GACTGTCGGTCGCGAGTATGACCGACAGTCCA GCACAGTGGCGGCCGCTCGAGT-30 and 50 -GACTGTCGGTCATACTCGC GACCGACAGTCGATATCTGCAGAATTCCACCAC-30 or 50 - GACTGCC GGTCGCGAGTATGACCGGCAGTCCAGCACAGTGGCGGCCGCTCGAGT-30 and 50 -GACTGCCGGTCATACTCGCGACCGGCAGTCGATATCTGCAGAATTCCACCAC-30 (recognition sequences are underlined, and the resulting overhanging base is in boldface and italicized) with polymerase incomplete primer extension site-directed mutagenesis [6,7] with Phusion High Fidelity Polymerase (all enzymes from New England Biolabs) using a lower primer concentration (0.2 lM instead of 0.5 lM) because primer dimer formation due to the presence of long complementary 50 overhangs led to no amplification. AhdI was selected because it cuts any sequence between its two recognition sequences and can be used for prolonged digestion without star activity. Silent site-directed mutagenesis [8,9] of the ampicillin resistance gene was also performed to remove an existing AhdI restriction site using primers T7 F and 50 -CGTAGTTATCTACACAACGGGGAGCCAGGCAACTATGG-30 . Vectors (3 lg) were digested with 10 U of AhdI (for single-nucleotide overhangs) or EcoRV HF (for blunt ends) for 16 h in CutSmart Buffer at 37 °C, incubated for a further 1 h with 10 U of Antarctic Phosphatase, and purified using a HiYield Gel/PCR DNA Mini Kit (Real Biotech). A 1.4-kb kanamycin resistance cassette from pUC18/Kan was prepared by PCR using phosphorylated primers pUC18-Kan F and pUC18-Kan R and Phusion High Fidelity Polymerase [7]. Purification and treatment of blunt product with dGTP is highly recommended for CG cloning because Taq strongly prefers to add a 30 A [3]. This is unlikely to require additional steps compared with TA cloning because proofreading
Notes & Tips / Anal. Biochem. 471 (2015) 80–82
81
Fig.1. Principles of CG cloning of a PCR product. A vector engineered to include two AhdI cleavage sites within the multiple cloning site (MCS) is digested to generate a linear fragment containing a single overhanging 30 C. Phosphatase treatment helps to eliminate empty vector background by preventing ligation of any singly cut vector or DNA, which loses the overhang over time. Blunt PCR product is either phosphorylated or prepared using phosphorylated primers, purified to remove dNTPs, and then treated with Taq DNA polymerase and dGTP to add single overhanging 30 Gs to the insert. Insert and vector are mixed and anneal via the single-base pairs. A nicked recombinant plasmid is then formed by T4 DNA ligase, which is transformed into Escherichia coli, which in this example will now be resistant to kanamycin due to the incorporation of the insert containing the KanR gene.
Fig.2. Direct comparison of TA, CG, and blunt-end cloning. See text for details. The average cloning efficiency is marked for clarity. Error bars are standard errors for the number of colonies for three independent experiments.
polymerases are routinely used for the initial amplification step to avoid errors. Gel-purified PCR product (500 ng) was treated with 200 lM dATP or dGTP using 5 U of Taq DNA polymerase for 30 min at 72 °C (or untreated for use in blunt-end ligation). Tailed product was column purified again to control for a possible inhibitory effect of dATP on ligation. Ligation used 50 ng each of insert and vector and 400 U of T4 DNA ligase at 15 °C for 30 min, 3 h, or 16 h. Next, 25-ll aliquots of XL10 Gold cells (Agilent) were transformed with 2.5 ll of ligation mix according to the manufacturer’s instructions. Cells were plated onto selective LB/agar. Cloning efficiency—the percentage of successful recombinants—was determined by first plating cells onto LB/agar containing ampicillin and then patching 26 clones onto kanamycin selective plates to confirm the presence of the KanR cassette. We also tried alternative strategies to prepare C-vector as previously used to create T-vectors [2]. Unacceptably low cloning efficiencies were obtained after attempts to add single overhanging Ts and Cs to blunt linearized vector using Taq polymerase or ddTTP/ ddCTP with terminal transferase, likely due to the poor tailing efficiency of Taq or the inability to phosphatase treat the vector, respectively. We directly compared TA, CG, and blunt-end cloning using the restriction-enzyme-treated vectors (Fig. 2). For shorter ligation times (30 min and 3 h), TA cloning performed poorly, yielding very low efficiencies (15 and 38%, respectively), although overnight ligation achieved useful efficiencies (74%). On the contrary, CG cloning achieved high efficiencies for all ligation times and approximately 4-fold more transformants than TA cloning. Thus, CG cloning can allow ligation and transformation on the same day and
with greater robustness than TA cloning. Blunt-end cloning performed the best, yielding close to 100% efficiency and more than 20-fold more colonies than TA cloning, reflecting the difficulty of ligating annealed single-base-pair overhangs. CG cloning is faster and more efficient than TA cloning, but for single genes blunt-end cloning using a proofreading polymerase is likely to be sufficient and most convenient because it does not require a tailing step and does not suffer from the high error rates observed using Taq DNA polymerase. However, none of these methods achieves directional cloning. Alternatively, ligation-independent cloning methods are simpler and more robust than cloning with restriction enzymes and ligation and achieve very high efficiencies with limited manipulations for a wide range of insert sizes [7], but they require the addition of long 50 overhangs to PCR primers, increasing the cost of cloning projects. However, for higher throughput cloning of short sequences for library construction, which do not tolerate the presence of concatemers, CG cloning is most suitable. During the preparation of this article, we became aware of commercially available kits developed by Lucigen using this principle [10], which the manufacturer refers to as ‘‘GC cloning,’’ with their own pSMART GC and pGC Blue vectors—and with additional steps to help eliminate empty vector background. As shown here in controlled experiments, CG cloning is indeed superior to TA cloning, and any C-vector can easily be prepared in-house. Acknowledgments The Brown Laboratory is supported by grants from the National Health and Medical Research Council (1008081) and the National
82
Notes & Tips / Anal. Biochem. 471 (2015) 80–82
Heart Foundation of Australia (G11S5757). The authors thank members of the Brown Laboratory for critically reviewing the manuscript. References [1] D.A. Mead, N.K. Pey, C. Herrnstadt, R.A. Marcil, L.M. Smith, A universal method for the direct cloning of PCR amplified nucleic acid, Biotechnology (N. Y.) 9 (1991) 657–663. [2] M.Y. Zhou, C.E. Gomez-Sanchez, Universal TA cloning, Curr. Issues Mol. Biol. 2 (2000) 1–7. [3] J.M. Clark, Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases, Nucleic Acids Res. 16 (1988) 9677–9686. [4] G. Hu, DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 30 end of a DNA fragment, DNA Cell Biol. 12 (1993) 763–770. [5] J.U. Jeung, S.K. Cho, K.S. Shim, S.H. Ok, D.S. Lim, J.S. Shin, Construction of two pGEM-7Zf(+) phagemid T-tail vectors using AhdI restriction endonuclease sites for direct cloning of PCR products, Plasmid 48 (2002) 160–163.
[6] H.E. Klock, E.J. Koesema, M.W. Knuth, S.A. Lesley, Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts, Proteins 71 (2008) 982–994. [7] J. Stevenson, J.R. Krycer, L. Phan, A.J. Brown, A practical comparison of ligationindependent cloning techniques, PLoS ONE 8 (2013) e83888. [8] J. Sanchis, L. Fernandez, J.D. Carballeira, J. Drone, Y. Gumulya, H. Hobenreich, D. Kahakeaw, S. Kille, R. Lohmer, J.J. Peyralans, J. Podtetenieff, S. Prasad, P. Soni, A. Taglieber, S. Wu, F.E. Zilly, M.T. Reetz, Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates, Appl. Microbiol. Biotechnol. 81 (2008) 387–397. [9] W.C. Tseng, J.W. Lin, T.Y. Wei, T.Y. Fang, A novel megaprimed and ligase-free, PCR-based, site-directed mutagenesis method, Anal. Biochem. 375 (2008) 376– 378. [10] D. A. Mead, R. Godiska, T. W. Schoenfeld, S. Hermanson, Plasmid comprising single stranded overhangs for use in replication of preferential gene sequences, patent US 7723103 BW, 2010.
