© 1999 Oxford University Press
Nucleic Acids Research, 1999, Vol. 27, No. 19
e26
Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites Daniel Tillett and Brett A. Neilan* School of Microbiology and Immunology, The University of New South Wales, Sydney 2052, Australia Received April 23, 1999; Revised and Accepted August 16, 1999
ABSTRACT We describe a simple method for the cloning of PCR products without the need for post-amplification enzymatic treatment. Tailed PCR primer sets are used to create complementary staggered overhangs on both insert and vector by a post-PCR denaturation– hybridisation reaction. The single-stranded overhangs are designed to allow directional cloning in a ligase-free manner. This ‘enzyme-free cloning’ procedure is highly efficient, and is not constrained by the need for the presence of suitable restriction enzyme sites within the plasmid vector. The avoidance of postamplification enzymatic procedures makes the technique rapid and reliable, avoiding the need for multiple sub-cloning steps. INTRODUCTION Numerous techniques have been developed for the cloning of PCR products. These include the incorporation of restriction enzyme sites into the PCR primers (1), blunt-end cloning (2,3), TA cloning (4,5), ligation independent cloning (LIC) (6–10) and in vivo cloning (11,12). While these methods are effective, they all require either extensive enzymatic treatment of the PCR product or vector (1,4–7,9,10,13), the use of PCR primers containing non-standard bases (8,13), or specialised vectors or bacterial strains (3,12). Under most circumstances these limitations pose little difficulty; however, under certain conditions the current techniques can be severely limiting. For example, the direct cloning of a PCR product into a particular site of an unusual expression vector is problematic. This can prove especially complex if the vector lacks suitable cloning sites, or a genetic basis for insert screening. Recently, a novel technique to clone PCR products, termed ‘hetero-stagger PCR cloning’, was introduced (14). This technique involves the generation of two related PCR products by the design of two sets of primers. The two sets are identical except the second primer set contains a 3 bp guanosine 5' tail (e.g. first set 5'-TAT . . ., second set 5'-GGGTAT . . .). Two PCR reactions are performed using the first untailed forward primer and the tailed reverse primer, and then the tailed forward primer and the untailed reverse primer. This produces two PCR products that, when mixed, denatured and allowed to reanneal, create fragments containing 3' CCC overhangs which are cloned by ligation to a vector containing 3' GGG overhangs. Although
elegant, this technique is limited by the need for extensive vector preparation and the requirement of a unique restriction enzyme site at the desired cloning location within the chosen vector. We reasoned that if the required 3 bp tail were increased to 12– 18 bp, then the resulting ‘hetero-staggered’ PCR products could be cloned using the LIC procedure (6). In addition, by PCR amplifying the vector using a compatible set of tailed and non-tailed primers, the PCR products could be cloned without further enzymatic reaction (Fig. 1). MATERIALS AND METHODS Primers were designed to amplify the cyanobacterial phycocyanin intergenic spacer region (PC-IGS) (15). Two parallel PCRs were performed using either PCβF (5'-GGCTGCTTGTTTACGCGACA-3') and PCαR-long (5'-GAACTAGGACATCCAGTACCACCAGCAACTAA-3'), or PCβF-long (5'-GGTACGGACTATGGCTGCTTGTTTACGCGACA-3') and PCαR (5'-CCAGTACCACCAGCAACTAA-3'). Both reactions contained 10 ng of Microcystis aeruginosa PCC7806 DNA and were subjected to 26 cycles of 94°C for 10 s, 50°C for 20 s and 72°C for 40 s, followed by a 7 min extension at 72°C using a high-fidelity PCR Taq/Pfu enzyme mix. The complementary linear vector was created by performing two parallel vector PCR using either Vup (5'-TAATCATGGTCATAGCTG-3') and Vdn-long (5'-ATGTCCTAGTTCACTGGCCGTCGTTTTAC-3'), or Vup-long (5'-ATAGTCCGTACCTAATCATGGTCATAGCTG-3') and Vdn (5'-ACTGGCCGTCGTTTTAC-3'). Each vector PCR contained 1 pg of pUC19 DNA and was subjected to 26 cycles of 94°C for 10 s, 50°C for 30 s and 72°C for 90 s, followed by a 7 min extension at 72°C. The two PC-IGS and pUC19 PCR products were pooled separately and excess primers removed using the Wizard PCR purification kit (Promega, Madison, WI). The denaturation and hybridisation reaction was performed using 50 ng of the pooled pUC PCR products and 12 ng of the PC-IGS PCR products (approximately equimolar) in 50 µl of hybridisation buffer (100 mM NaCl, 10 mM Tris–HCl, pH 7.4, 1 mM EDTA, pH 8). A control hybridisation reaction containing 50 ng of the pooled vector amplicons alone was also performed. Both hybridisation reactions were heated to 95°C for 3 min then subjected to four annealing cycles of 65°C for 2 min and 25°C for 15 min. Two microlitres of each reaction were used to transform Escherichia coli DH5α CaCl2 competent cells (16). The resulting colonies were screened for the presence of the cloned PC-IGS PCR product by alkaline lysis plasmid screening and colony PCR using the PC-IGS primers (15,16).
*To whom correspondence should be addressed. Tel: +61 2 9385 3235; Fax: +61 2 9385 1591; Email:
[email protected]
e26
Nucleic Acids Research, 1999, Vol. 27, No. 19
RESULTS AND DISCUSSION
Figure 1. The enzyme-free cloning strategy. Four PCR reactions are performed, two vector and two insert: the first vector PCR is performed with the 5' vector short and the 3' vector long (tailed) primers, while the second vector PCR uses the 5' vector long and the 3' vector short primers; the first insert PCR uses the 5' insert short and 3' insert long primers, while the second is performed using the 5' insert long and the 3' insert short primers. The long primers differ from the short primers by possessing a 12–15 bp 5' tail, with each of the two nonhomologous insert primer tails complementary to only one of the two vector primer tails, thus ensuring the directional cloning of the desired fragment. Equimolar volumes of the four PCR products are mixed, heat-denatured and allowed to re-anneal. Eight annealing products of equal probability are created, of which four contain either 5' or 3' 12–18 bp overhangs. Low temperature annealing (25°C) allows these four products to form two nicked circular products which can be transformed without ligation.
The enzyme-free procedure provided a highly efficient means of cloning PCR products independent of vector restriction enzyme sites (Table 1). Theoretically, 50% of the PCR products should be cloneable, that is, all molecules with either 5' or 3' overhangs. In practice, transformation efficiencies of up to 8 × 104/µg of insert DNA were obtained using standard E.coli CaCl2 competent cells (106/µg of pUC19 plasmid), with all of the screened colonies containing the correct PC-IGS insert (Table 1). The use of a high-fidelity PCR mix minimises the introduction of PCR-derived mutations, both within the insert and vector. In addition, no colonies were obtained from the transformation of the control PCR amplified linear vector hybridisation reaction. The enzyme-free cloning approach has a number of advantages over other methods. Firstly, no post PCR enzyme reactions are required (e.g. restriction enzyme digestions, phosphatase treatments, partial DNase digestions or ligations), thus reducing the potential for cloning failure. Secondly, this approach is simple and rapid, requiring minimal handling. Thirdly, this procedure enables the PCR insert to be cloned directly into the final vector independent of restriction enzyme site. This is particularly useful for ensuring a single insert fragment is cloned into the correct reading frame for protein expression, or the creation of fusion proteins. In addition, the technique can be used to easily introduce a short amino acid tag sequence into either the N- or C-terminus of a protein, for example, this technique has been used to create a number of overexpressed fusion protein containing both N- and C-terminal six histidine affinity purification tags (unpublished data). Finally, because of the initial success obtained using this procedure we have not attempted to optimise this protocol further; however, we have found the use of a cycled reannealing step is critical in ensuring efficient formation of the tailed products. While this enzyme-free cloning approach does provide a number of advantages over other cloning strategies, it does have minor limitations. Firstly, this approach requires a number of primers (eight); however, we believe the advantages offered by this technique outweigh the added cost, especially given the low cost of commercial oligonucleotides. We have found for many applications a common set of vector primers can be used and thus only the two-tailed insert primers are required over other PCR cloning procedures. Secondly, this approach requires that both the vector and insert are able to be amplified by PCR. Zhang et al. (17) have recently introduced a procedure based on homologous recombination (termed ET cloning) able to overcome this limitation. However, this procedure
Table 1. Cloning of the PC-IGS region from M.aeruginosa PCC 7806 using the enzyme-free cloning method Vector
Insert
Linear pUC19 PCR (50 ng)
PC-IGS PCR (12.5 ng)
Coloniesa 40
Percent with insertb 100
Linear pUC19 PCR (50 ng)
–
0
–
Circular pUC19 plasmid (1 ng)
–
1020
–
colonies obtained from transformation with 2 µl of the hybridisation reaction (50 µl total) using 150 µl of CaCl2 competent E.coli DH5α cells. bAs determined from 18 randomly selected colonies by alkaline lysis plasmid miniprep and PC-IGS PCR (15,16). aAmpicillin-resistant
ii
Nucleic Acids Research, 1999, Vol. 27, No. 19
requires two rounds of transformation and selection and either the use of specialised E.coli strains (sbcA), or the use of a second modifying plasmid (pBAD-ETλ). While we believe that the ET cloning procedure is superior for some applications (e.g. the engineering of very large BAC clones), the simplicity of the described enzyme-free cloning procedure should prove advantageous in many applications. ACKNOWLEDGEMENTS D.T. would like thank Carolina Beltran, Torsten Thomas, Canan Sinan and Paul March for their support and encouragement. This work was financially supported by the Australian Research Council and the CRC for Water Quality and Treatment. REFERENCES 1. Scharf,S.J., Horn,G.T. and Erlich,H.A. (1986) Science, 233, 1076–1078. 2. Scharf,S.J. (1990) In Innis,M.A., Gelfand,D.H., Sninsky,J.J. and White,T.J. (eds), PCR Protocols. Academic Press, New York, NY, pp. 84–91.
e26
3. Costa,G.L., Grafsky,A. and Weiner,M.P. (1994) PCR Methods Appl., 3, 338–345. 4. Holton,T.A. and Graham,M.W. (1991) Nucleic Acids Res., 19, 1156. 5. Marchuk,D., Drumm,M., Saulino,A. and Collins,F.S. (1991) Nucleic Acids Res., 19, 1154. 6. Aslanidis,C. and de Jong,P.J. (1990) Nucleic Acids Res., 20, 6069–6074. 7. Shuldiner,A.R., Scott,L.A. and Roth,J. (1990) Nucleic Acids Res., 18, 1920. 8. Kaluz,S. and Flint,A.P.F. (1994) Nucleic Acids Res., 22, 4845. 9. Yang,Y.-S., Watson,W.J., Tucker,P.W. and Capra,J.D. (1993) Nucleic Acids Res., 21, 1889–1893. 10. Hsiao,K. (1993) Nucleic Acids Res., 21, 5528–5529. 11. Bubeck,P., Winkler,M. and Bautsch,W. (1993) Nucleic Acids Res., 21, 3601–3602. 12. Oliner,J.D., Kinzer,K.W. and Vogelstein,B. (1993) Nucleic Acids Res., 21, 5192–5197. 13. Nisson,P., Rashtchian,A. and Watkins,P. (1991) PCR Methods Appl., 1, 120–123. 14. Liu,Z. (1996) Nucleic Acids Res., 24, 2458–2459. 15. Neilan,B.A., Jacobs,D. and Goodman,A.E. (1995) Appl. Environ. Microbiol., 61, 3875–3883. 16. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 17. Zhang,Y., Buchholz,F., Muyrers,J.P.P. and Stewart,A.F. (1998) Nature Genet., 20, 123–128.
iii
