- Email: [email protected]
A modified version of the digestion–ligation cloning method for more efficient molecular cloning
YABIO 11670
No. of Pages 3, Model 5G
21 March 2014 Analytical Biochemistry xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
2
Notes & Tips
6 4 7 5
A modified version of the digestion–ligation cloning method for more efficient molecular cloning
8
Song Gao, Yanling Li, Jiannan Zhang, Hongman Chen, Daming Ren, Lijun Zhang, Yingfeng An ⇑
9
College of Biosciences and Biotechnology, Shenyang Agricultural University, Shenyang 110161, China
10 1 4 2 2 13 14 15 16 17 18 19 20 21 22 23
a r t i c l e
i n f o
Article history: Received 1 February 2014 Received in revised form 25 February 2014 Accepted 26 February 2014 Available online xxxx Keywords: Blunt-end DNA ligation T4 DNA ligase Molecular cloning Digestion–ligation approach
a b s t r a c t Here we describe a modified version of the digestion–ligation approach for efficient molecular cloning. In comparison with the original method, the modified method has the additional steps of gel purification and a second ligation after the first ligation of the linearized vector and DNA insert. During this process, the efficiency and reproducibility could be significantly improved for both stick-end cloning and bluntend cloning. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology. Ó 2014 Elsevier Inc. All rights reserved.
25 26 27 28 29 30 31 32
33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
T4 DNA ligase-dependent DNA ligation is an important tool in molecular biology, molecular genetics, biochemistry, and the like. Sticky-end DNA ligation and blunt-end DNA ligation are both commonly used strategies [1]. However, commonly blunt-end ligation has much lower efficiency when compared with sticky-end DNA ligation, and self-ligation of vector often occurs. Although various parameters having effects on the efficiency of blunt-end DNA ligation (e.g., temperature, components of buffer, molecular weight of the DNA fragments) have been studied and optimized in some laboratories [2,3], the efficiency of ligation is still far from satisfactory. As a result, commonly screening of positive clones after ligation is laborious and time-consuming. Although theoretically DNA fragments could also be amplified by polymerase chain reaction (PCR)1 and cloned by some PCR-dependent methods (e.g., TA cloning, ligation-independent cloning, Gateway cloning, PCR-based construction of complete recombinant plasmids, Gibson assembly cloning) [4–14], in some cases the classical digestion–ligation strategy should be more convenient, for example, releasing a fragment from a vector by restriction digestion followed by subcloning into another vector. When subcloning is performed with the digestion– ligation approach instead of these PCR-dependent methods, design and synthesis of primers are not required and, more important, random mutations would not be introduced. In addition, some DNA fragments (e.g., DNA fragments originated from genome DNAs) could not be amplified by PCR because of their large sizes or high ⇑ Corresponding author. 1
E-mail address: [email protected] (Y. An). Abbreviation used: PCR, polymerase chain reaction.
GC (guanine–cytosine) contents. Therefore, although multiple PCR-dependent cloning approaches have been developed, and each has proven to be successful for many applications, the classical digestion–ligation method is still the preferred method in many laboratories all over the world. However, in many cases even the efficiency of sticky-end DNA ligation still needs to be improved. In these cases, researchers might need to optimize various parameters (e.g., reaction temperature, components of buffer, molar ratio of vector/insert) before obtaining success. Therefore, a modified version of the classical cloning method with improved efficiency should be valuable. In this study, we have developed a modified approach for cloning of restriction digested fragments that is robust, more efficient, and especially suitable for cloning of blunt-end DNAs. A 1.3-kb DNA insert was released from pLYL01 plasmid with the restriction endonuclease SmaI and purified from a 1% agarose gel by an AxyPrep DNA Gel Extraction Kit (Axygen Bio Sciences, Union City, CA, USA). In separate experiments, pUC19 plasmid was also digested with SmaI and purified by a PCR purification kit (Axygen Bio Sciences) to give a 2.4-kb blunt-end vector. Then the bluntend insert and vector were mixed and ligated with a T4 DNA Ligation Kit (version 2.1, TaKaRa Biotechnology, Dalian, China) in a total volume of 100 ll. The concentration of the linearized vector was fixed to be 2 lmol/L, and different concentrations of DNA inserts (1:1, 1:3, and 3:1 for molar ratios of vector/insert) were used for ligation. As a control, either 2 lmol/L linearized insert or 2 lmol/L blunt-end vector was performed with self-ligation. After ligation at 37 °C for 30 min, the ligation products of insert and vector mixtures were performed with agarose gel electrophoresis, and the fragments with the expected size of DNA insert + vector (i.e.,
http://dx.doi.org/10.1016/j.ab.2014.02.031 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: S. Gao et al., A modified version of the digestion–ligation cloning method for more efficient molecular cloning, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.031
60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88
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No. of Pages 3, Model 5G
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A modified version of the digestion–ligation cloning method for more efficient molecular cloning / S. Gao et al. / Anal. Biochem. xxx (2014) xxx–xxx
3.7 kb) were purified by an AxyPrep DNA Gel Extraction Kit. The purified DNAs were performed with a second DNA ligation at 37 °C for 30 min, followed by DNA purification with a PCR cleanup kit. The purified ligation products were transformed into Escherichia coli JM109 by electroporation and plated on LB agar plates supplemented with ampicillin. After overnight growth at 37 °C, 30 transformants were picked up for colony PCR with primers pUC19-For (50 -GCGGG TGTTG GCGGG TGTCG G-30 ) and pUC19Rev (50 -CTGGA AAGCG GGCAG TGAGC GCAAC-30 ) annealing to vector. The colony PCR products above were analyzed by agarose gel electrophoresis. Then recombinant plasmids extracted from positive clones were submitted to DNA sequencing. As a control, the first ligation products with different concentrations of DNA inserts (1:1, 1:3, and 3:1 for molar ratios of vector/insert) were directly transformed into E. coli, and 30 transformants after each transformation were picked up for colony PCR with primers pUC19-For and pUC19-Rev and the colony PCR products were analyzed by agarose gel electrophoresis. The comparison of the modified digestion–ligation method and its original version are illustrated in Fig. 1. For both methods, vector and DNA insert are prepared by restriction digestion (step 1) and DNA ligation is performed with T4 DNA ligase (step 2). For the modified version of the method, the target DNA fragment with the expected size of DNA insert + vector in the ligation mixture is size selected by gel purification (step 3), followed by self-ligation (step 4) and transformation into E. coli (step 5), whereas for the original version of the method, the mixture of the first ligation product is directly transformed into E. coli (step 30 ), followed by a screening stage to identify the positive construct (step 40 ). According to our experience, for unexpected reasons, the incomplete digestion of vector could not be avoided even with a high concentration of enzymes and long digestion time (Fig. 1, step 1). Fortunately, these fragments originating from incomplete digestion of vector could be eliminated by the gel purification step (Fig. 1, step 30 ). In addition, commonly a strong possibility of undesired ligation (e.g., inverted and tandem repeat of vector, self-circularization of vector) during T4 DNA ligase-dependent cloning is inevitable
(Fig. 1, step 2, and Fig. 2, lanes 3–6). Consequently, only a small fraction of the ligation product may introduce the correct construction, especially when a ligation reaction is challenging (e.g., most blunt-end DNA ligation reactions, some sticky-end ligation reactions). In these cases, the screening of positive clones should be laborious and time-consuming. However, with this modified cloning method, the full-length ligation product can be isolated by gel purification. This enrichment and purification process would effectively eliminate the interference of DNAs with undesired ligation and avoid the workload of the subsequent screening step. In addition, according to the design of this modified method, only one DNA terminus of the DNA insert needs to be ligated with vector after each ligation step, whereas according to original version of the method, both termini of the DNA insert need to be ligated with vector after a single ligation step. It is obvious that the modified method should be less challenging, which may contribute to its higher efficiency and reproducibility. The robustness and effectiveness of this method were tested by applying it to cloning of a blunt-end DNA. The mixtures of vector and DNA insert with different molar ratios (1:1, 1:3, and 3:1) were performed with DNA ligation, and the ligation products were analyzed by agarose gel electrophoresis. Although the desired bands of vector + DNA insert with the expected size (i.e., 3.7 kb) were obtained from all three ligation reactions (Fig. 2, lanes 4–6), the yields were obviously different. This is consistent with the fact that the concentrations of DNA fragments and molar ratios of vector/inserts obviously can have an effect on the DNA ligation [15]. Fortunately, this kind of effect on the first ligation can be eliminated by the enrichment and concentration functions of the gel purification step, which may also contribute to the satisfactory efficiency and reproducibility of this modified method. In a typical experiment, 1.5 103 transformants were obtained after 1 ng of DNA from the first gel purification being performed with a second ligation, purification, and transformation into E. coli by electroporation. The randomly selected transformants were picked up for colony PCR to show that the majority of the clones (27/30) were positive clones, which was further proved by DNA
Fig.1. Comparison of the modified digestion–ligation method and its original version. For both methods, vector and DNA insert are prepared by restriction digestion (step 1) and DNA ligation is performed with T4 DNA ligase (step 2). For the modified version of the method, the target DNA fragment with the expected size of DNA insert + vector in the ligation mixture is size selected by gel purification (step 3), followed by intramolecular ligation (step 4) and transformation into E. coli, to provide circular recombinant plasmid DNA (step 5), whereas for the original version of the method, the mixture of the first ligation product is directly transformed into E. coli (step 30 ), followed by a screening stage to identify the positive construct (step 40 ).
Please cite this article in press as: S. Gao et al., A modified version of the digestion–ligation cloning method for more efficient molecular cloning, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.031
126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162
YABIO 11670
No. of Pages 3, Model 5G
21 March 2014 A modified version of the digestion–ligation cloning method for more efficient molecular cloning / S. Gao et al. / Anal. Biochem. xxx (2014) xxx–xxx
Fig.2. Agarose gel showing the products obtained by first ligation of blunt-end vector and insert. Lane M: DNA ladder; lane 1: linear vector; lane 2: DNA insert; lane 3: self-ligation of linear vector; lanes 4, 5, and 6: ligation products of vector and DNA insert with molar ratios of 1:1, 1:3, and 3:1, respectively. The bands of expected fragments in the ligation mixtures are boxed.
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sequencing, and only a minority of the clones (3/30) were negative clones originating from self-ligation of vector. Although theoretically self-ligation of vector could be avoided, it occurred for some unexpected reasons. Fortunately, self-ligation of vector occurred with only low frequency and could not significantly affect the cloning efficiency. As a control, the first ligation products of vector and DNA insert with different molar ratios (1:1, 1:3, and 3:1) were directly transformed into E. coli. In total, 90 randomly selected transformants after three transformations were picked up for colony PCR to show that all of the clones were self-ligation of vectors that might have originated from incomplete digestion of vector (Fig. 1, step 1). Therefore, although the modified method would need more time (1.5 h longer) than the classical method because of the additional two steps (i.e., gel purification and second ligation) (Fig. 1, steps 30 and 40 ), the cloning efficiency and reproducibility were significantly improved and the time-consuming screening steps (taking no less than 3 h; Fig. 1, step 4) could be avoided as compensation. In fact, in some cases, even the ligation of sticky-end DNAs may encounter difficulties. For instance, in our laboratory, levansucrase genes BS-sucB, BA-sucB1, and CA-sucB from Bacillus subtilis, Bacillus amyloliquefaciens, and Clostridium acetobutylicum, respectively, were performed with sticky-end ligation with pETM 11 vector. By using the classical digestion–ligation method, no clone was constructed even after three rounds of digestion–ligation with optimized reaction conditions, whereas by using the modified method, all of the clones were successfully constructed without optimization of the reaction conditions (data not shown). Although we believe that by using the classical method the clones could be definitely constructed after further optimization of the reaction conditions, the modified method seems to be more robust, at least in our hands.
3
In conclusion, we have established a modified restriction ligation method that is helpful for improving cloning efficiency, especially for cloning of blunt-end DNA inserts. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology.
194
Acknowledgments
199
This work was supported by the National Natural Science Foundations of China (31100045 and 31270114) and the Program for Liaoning Excellent Talents in University (LJQ2011067).
200
References
203
[1] T. Conze, A. Shetye, Y. Tanaka, J. Gu, C. Larsson, J. Göransson, G. Tavoosidana, O. Söderberg, M. Nilsson, U. Landegren, Analysis of genes, transcripts, and proteins via DNA ligation, Annu. Rev. Anal. Chem. 2 (2009) 215–239. [2] F. Gao, H. Zhou, W. Li, X. Zhang, Detection of ligation products of DNA linkers with 50 -OH ends by denaturing PAGE silver stain, PLoS One 7 (2012) e39251. [3] Y. Yoshino, M. Ishida, A. Horii, A new 10-min ligation method using a modified buffer system with a very low amount of T4 DNA ligase: the coffee break ligation technique, Biotechnol. Lett. 29 (2007) 1557–1560. [4] J.L. Hartley, G.F. Temple, M.A. Brasch, DNA cloning using in vitro site-specific recombination, Genome Res. 10 (2000) 1788–1795. [5] T. Unger, Y. Jacobovitch, A. Dantes, R. Bernheim, Y. Peleg, Applications of the restriction free (RF) cloning procedure for molecular manipulations and protein expression, J. Struct. Biol. 172 (2010) 34–44. [6] J.Y. Jeong, H.S. Yim, J.Y. Ryu, H.S. Lee, J.H. Lee, D.S. Seen, S.G. Kang, One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies, Appl. Environ. Microbiol. 78 (2012) 5440–5443. [7] C.R. Irwin, A. Farmer, D.O. Willer, D.H. Evans, In-Fusion cloning with vaccinia virus DNA polymerase, Methods Mol. Biol. 890 (2012) 23–35. [8] D.G. Gibson, L. Young, R.Y. Chuang, J.C. Venter, C.A. Hutchison 3rd, H.O. Smith, Enzymatic assembly of DNA molecules up to several hundred kilobases, Nat. Methods 6 (2009) 343–345. [9] R.B. Yang, L.J. Bi, X.E. Zhang, A novel T-type overhangs improve the enzymefree cloning of PCR products, Mol. Biotechnol. 55 (2013) 10–16. [10] H.E. Klock, S.A. Lesley, The polymerase incomplete primer extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis, Methods Mol. Biol. 498 (2009) 91–103. [11] C. Li, A. Wen, B. Shen, J. Lu, Y. Huang, Y. Chang, FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method, BMC Biotechnol. 11 (2011) 92. [12] D. Zheng, X. Liu, Y. Zhou, 3GC cloning: PCR products cloning mediated by terminal deoxynucleotidyl transferase, Anal. Biochem. 378 (2008) 108–110. [13] M. Blanusa, A. Schenk, H. Sadeghi, J. Marienhagen, U. Schwaneberg, Phosphorothioate-based ligase-independent gene cloning (PLICing): an enzyme-free and sequence-independent cloning method, Anal. Biochem. 406 (2010) 141–146. [14] T. Baumann, K.M. Arndt, K.M. Müller, Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V, BMC Biotechnol. 13 (2013) 81. [15] R.M. Cranenburgh, An equation for calculating the volumetric ratios required in a ligation reaction, Appl. Microbiol. Biotechnol. 65 (2004) 200–202.
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Please cite this article in press as: S. Gao et al., A modified version of the digestion–ligation cloning method for more efficient molecular cloning, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.031
No. of Pages 3, Model 5G
21 March 2014 Analytical Biochemistry xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
2
Notes & Tips
6 4 7 5
A modified version of the digestion–ligation cloning method for more efficient molecular cloning
8
Song Gao, Yanling Li, Jiannan Zhang, Hongman Chen, Daming Ren, Lijun Zhang, Yingfeng An ⇑
9
College of Biosciences and Biotechnology, Shenyang Agricultural University, Shenyang 110161, China
10 1 4 2 2 13 14 15 16 17 18 19 20 21 22 23
a r t i c l e
i n f o
Article history: Received 1 February 2014 Received in revised form 25 February 2014 Accepted 26 February 2014 Available online xxxx Keywords: Blunt-end DNA ligation T4 DNA ligase Molecular cloning Digestion–ligation approach
a b s t r a c t Here we describe a modified version of the digestion–ligation approach for efficient molecular cloning. In comparison with the original method, the modified method has the additional steps of gel purification and a second ligation after the first ligation of the linearized vector and DNA insert. During this process, the efficiency and reproducibility could be significantly improved for both stick-end cloning and bluntend cloning. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology. Ó 2014 Elsevier Inc. All rights reserved.
25 26 27 28 29 30 31 32
33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
T4 DNA ligase-dependent DNA ligation is an important tool in molecular biology, molecular genetics, biochemistry, and the like. Sticky-end DNA ligation and blunt-end DNA ligation are both commonly used strategies [1]. However, commonly blunt-end ligation has much lower efficiency when compared with sticky-end DNA ligation, and self-ligation of vector often occurs. Although various parameters having effects on the efficiency of blunt-end DNA ligation (e.g., temperature, components of buffer, molecular weight of the DNA fragments) have been studied and optimized in some laboratories [2,3], the efficiency of ligation is still far from satisfactory. As a result, commonly screening of positive clones after ligation is laborious and time-consuming. Although theoretically DNA fragments could also be amplified by polymerase chain reaction (PCR)1 and cloned by some PCR-dependent methods (e.g., TA cloning, ligation-independent cloning, Gateway cloning, PCR-based construction of complete recombinant plasmids, Gibson assembly cloning) [4–14], in some cases the classical digestion–ligation strategy should be more convenient, for example, releasing a fragment from a vector by restriction digestion followed by subcloning into another vector. When subcloning is performed with the digestion– ligation approach instead of these PCR-dependent methods, design and synthesis of primers are not required and, more important, random mutations would not be introduced. In addition, some DNA fragments (e.g., DNA fragments originated from genome DNAs) could not be amplified by PCR because of their large sizes or high ⇑ Corresponding author. 1
E-mail address: [email protected] (Y. An). Abbreviation used: PCR, polymerase chain reaction.
GC (guanine–cytosine) contents. Therefore, although multiple PCR-dependent cloning approaches have been developed, and each has proven to be successful for many applications, the classical digestion–ligation method is still the preferred method in many laboratories all over the world. However, in many cases even the efficiency of sticky-end DNA ligation still needs to be improved. In these cases, researchers might need to optimize various parameters (e.g., reaction temperature, components of buffer, molar ratio of vector/insert) before obtaining success. Therefore, a modified version of the classical cloning method with improved efficiency should be valuable. In this study, we have developed a modified approach for cloning of restriction digested fragments that is robust, more efficient, and especially suitable for cloning of blunt-end DNAs. A 1.3-kb DNA insert was released from pLYL01 plasmid with the restriction endonuclease SmaI and purified from a 1% agarose gel by an AxyPrep DNA Gel Extraction Kit (Axygen Bio Sciences, Union City, CA, USA). In separate experiments, pUC19 plasmid was also digested with SmaI and purified by a PCR purification kit (Axygen Bio Sciences) to give a 2.4-kb blunt-end vector. Then the bluntend insert and vector were mixed and ligated with a T4 DNA Ligation Kit (version 2.1, TaKaRa Biotechnology, Dalian, China) in a total volume of 100 ll. The concentration of the linearized vector was fixed to be 2 lmol/L, and different concentrations of DNA inserts (1:1, 1:3, and 3:1 for molar ratios of vector/insert) were used for ligation. As a control, either 2 lmol/L linearized insert or 2 lmol/L blunt-end vector was performed with self-ligation. After ligation at 37 °C for 30 min, the ligation products of insert and vector mixtures were performed with agarose gel electrophoresis, and the fragments with the expected size of DNA insert + vector (i.e.,
http://dx.doi.org/10.1016/j.ab.2014.02.031 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: S. Gao et al., A modified version of the digestion–ligation cloning method for more efficient molecular cloning, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.031
60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88
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No. of Pages 3, Model 5G
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A modified version of the digestion–ligation cloning method for more efficient molecular cloning / S. Gao et al. / Anal. Biochem. xxx (2014) xxx–xxx
3.7 kb) were purified by an AxyPrep DNA Gel Extraction Kit. The purified DNAs were performed with a second DNA ligation at 37 °C for 30 min, followed by DNA purification with a PCR cleanup kit. The purified ligation products were transformed into Escherichia coli JM109 by electroporation and plated on LB agar plates supplemented with ampicillin. After overnight growth at 37 °C, 30 transformants were picked up for colony PCR with primers pUC19-For (50 -GCGGG TGTTG GCGGG TGTCG G-30 ) and pUC19Rev (50 -CTGGA AAGCG GGCAG TGAGC GCAAC-30 ) annealing to vector. The colony PCR products above were analyzed by agarose gel electrophoresis. Then recombinant plasmids extracted from positive clones were submitted to DNA sequencing. As a control, the first ligation products with different concentrations of DNA inserts (1:1, 1:3, and 3:1 for molar ratios of vector/insert) were directly transformed into E. coli, and 30 transformants after each transformation were picked up for colony PCR with primers pUC19-For and pUC19-Rev and the colony PCR products were analyzed by agarose gel electrophoresis. The comparison of the modified digestion–ligation method and its original version are illustrated in Fig. 1. For both methods, vector and DNA insert are prepared by restriction digestion (step 1) and DNA ligation is performed with T4 DNA ligase (step 2). For the modified version of the method, the target DNA fragment with the expected size of DNA insert + vector in the ligation mixture is size selected by gel purification (step 3), followed by self-ligation (step 4) and transformation into E. coli (step 5), whereas for the original version of the method, the mixture of the first ligation product is directly transformed into E. coli (step 30 ), followed by a screening stage to identify the positive construct (step 40 ). According to our experience, for unexpected reasons, the incomplete digestion of vector could not be avoided even with a high concentration of enzymes and long digestion time (Fig. 1, step 1). Fortunately, these fragments originating from incomplete digestion of vector could be eliminated by the gel purification step (Fig. 1, step 30 ). In addition, commonly a strong possibility of undesired ligation (e.g., inverted and tandem repeat of vector, self-circularization of vector) during T4 DNA ligase-dependent cloning is inevitable
(Fig. 1, step 2, and Fig. 2, lanes 3–6). Consequently, only a small fraction of the ligation product may introduce the correct construction, especially when a ligation reaction is challenging (e.g., most blunt-end DNA ligation reactions, some sticky-end ligation reactions). In these cases, the screening of positive clones should be laborious and time-consuming. However, with this modified cloning method, the full-length ligation product can be isolated by gel purification. This enrichment and purification process would effectively eliminate the interference of DNAs with undesired ligation and avoid the workload of the subsequent screening step. In addition, according to the design of this modified method, only one DNA terminus of the DNA insert needs to be ligated with vector after each ligation step, whereas according to original version of the method, both termini of the DNA insert need to be ligated with vector after a single ligation step. It is obvious that the modified method should be less challenging, which may contribute to its higher efficiency and reproducibility. The robustness and effectiveness of this method were tested by applying it to cloning of a blunt-end DNA. The mixtures of vector and DNA insert with different molar ratios (1:1, 1:3, and 3:1) were performed with DNA ligation, and the ligation products were analyzed by agarose gel electrophoresis. Although the desired bands of vector + DNA insert with the expected size (i.e., 3.7 kb) were obtained from all three ligation reactions (Fig. 2, lanes 4–6), the yields were obviously different. This is consistent with the fact that the concentrations of DNA fragments and molar ratios of vector/inserts obviously can have an effect on the DNA ligation [15]. Fortunately, this kind of effect on the first ligation can be eliminated by the enrichment and concentration functions of the gel purification step, which may also contribute to the satisfactory efficiency and reproducibility of this modified method. In a typical experiment, 1.5 103 transformants were obtained after 1 ng of DNA from the first gel purification being performed with a second ligation, purification, and transformation into E. coli by electroporation. The randomly selected transformants were picked up for colony PCR to show that the majority of the clones (27/30) were positive clones, which was further proved by DNA
Fig.1. Comparison of the modified digestion–ligation method and its original version. For both methods, vector and DNA insert are prepared by restriction digestion (step 1) and DNA ligation is performed with T4 DNA ligase (step 2). For the modified version of the method, the target DNA fragment with the expected size of DNA insert + vector in the ligation mixture is size selected by gel purification (step 3), followed by intramolecular ligation (step 4) and transformation into E. coli, to provide circular recombinant plasmid DNA (step 5), whereas for the original version of the method, the mixture of the first ligation product is directly transformed into E. coli (step 30 ), followed by a screening stage to identify the positive construct (step 40 ).
Please cite this article in press as: S. Gao et al., A modified version of the digestion–ligation cloning method for more efficient molecular cloning, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.031
126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162
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No. of Pages 3, Model 5G
21 March 2014 A modified version of the digestion–ligation cloning method for more efficient molecular cloning / S. Gao et al. / Anal. Biochem. xxx (2014) xxx–xxx
Fig.2. Agarose gel showing the products obtained by first ligation of blunt-end vector and insert. Lane M: DNA ladder; lane 1: linear vector; lane 2: DNA insert; lane 3: self-ligation of linear vector; lanes 4, 5, and 6: ligation products of vector and DNA insert with molar ratios of 1:1, 1:3, and 3:1, respectively. The bands of expected fragments in the ligation mixtures are boxed.
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sequencing, and only a minority of the clones (3/30) were negative clones originating from self-ligation of vector. Although theoretically self-ligation of vector could be avoided, it occurred for some unexpected reasons. Fortunately, self-ligation of vector occurred with only low frequency and could not significantly affect the cloning efficiency. As a control, the first ligation products of vector and DNA insert with different molar ratios (1:1, 1:3, and 3:1) were directly transformed into E. coli. In total, 90 randomly selected transformants after three transformations were picked up for colony PCR to show that all of the clones were self-ligation of vectors that might have originated from incomplete digestion of vector (Fig. 1, step 1). Therefore, although the modified method would need more time (1.5 h longer) than the classical method because of the additional two steps (i.e., gel purification and second ligation) (Fig. 1, steps 30 and 40 ), the cloning efficiency and reproducibility were significantly improved and the time-consuming screening steps (taking no less than 3 h; Fig. 1, step 4) could be avoided as compensation. In fact, in some cases, even the ligation of sticky-end DNAs may encounter difficulties. For instance, in our laboratory, levansucrase genes BS-sucB, BA-sucB1, and CA-sucB from Bacillus subtilis, Bacillus amyloliquefaciens, and Clostridium acetobutylicum, respectively, were performed with sticky-end ligation with pETM 11 vector. By using the classical digestion–ligation method, no clone was constructed even after three rounds of digestion–ligation with optimized reaction conditions, whereas by using the modified method, all of the clones were successfully constructed without optimization of the reaction conditions (data not shown). Although we believe that by using the classical method the clones could be definitely constructed after further optimization of the reaction conditions, the modified method seems to be more robust, at least in our hands.
3
In conclusion, we have established a modified restriction ligation method that is helpful for improving cloning efficiency, especially for cloning of blunt-end DNA inserts. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology.
194
Acknowledgments
199
This work was supported by the National Natural Science Foundations of China (31100045 and 31270114) and the Program for Liaoning Excellent Talents in University (LJQ2011067).
200
References
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Please cite this article in press as: S. Gao et al., A modified version of the digestion–ligation cloning method for more efficient molecular cloning, Anal. Biochem. (2014), http://dx.doi.org/10.1016/j.ab.2014.02.031