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A simple method for directional multimerization of DNA sequences
Technical Tips Online, Vol. 2, 1997
A simple method for directional multimerization of DNA sequences Pieter B.F. Ouwerkerk and Johan Memelink Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, PO Box 9505, 2300 RA Leiden, The Netherlands
Keywords: Cloning & sequencing ▼We describe a simple method for the multimerization of DNA sequences in a directional manner. Multimers have a number of applications, especially in gene promoter analysis and for cloning of DNA-binding proteins using southwestern screening or the yeast one-hybrid system. Multimerization of a DNA sequence is usually performed by concatenation of synthetic oligonucleotides and subsequent cloning. Some strategies for directional multimerization have been described (Ref. 1, 2). However, we found that the considerable number of synthesis errors in many oligonucleotide preparations makes the selection of the correct multimer a laborious task. In addition, the yield of longer multimers in concatenation reactions is low, making the cloning of large multimers technically troublesome. Our method takes advantage of the polylinker sequences of the pIC vector series (Ref. 3). The DNA sequence of interest is cloned such that it is flanked by enzyme sites that generate compatible ends, which upon ligation are not recognized by either enzyme. The pIC vectors offer the compatible sticky-end combinations BamHI/BglII and SalI/XhoI. The example described here shows dimerization using BamHI/BglII (Fig. 1). The monomer fragment is excised with PstI/BglII and reintroduced between the PstI and BamHI sites of the monomer-containing plasmid. The resulting plasmid contains a head-to-tail dimer. Repeating the procedure with the dimer-containing plasmid results in a tetramer, and so on. Because multimerization proceeds
exponentially according to the formula 2n where n is the number of cloning steps starting with the monomer, large multimers are obtained after relatively few cloning steps. The monomer sequences are separated by the 6-bp fusions of the compatible restriction enzyme sites. In addition to BamHI/BgIII and SalI/XhoI fragments, any fragment can be cloned blunt-ended using the EcoRV and SmaI sites between the BamHI and BglII sites in the pIC-19H and 20H plasmids. In this case, multimerization results in monomers separated by 12 bp instead of the 6-bp BamHI/BglII or SalI/XhoI fusions. Besides its simplicity, a clear advantage of our method is that it starts with a monomer the sequence of which can be verified prior to the multimerization procedure. The method is not only applicable to short synthetic oligonucleotides, but also to larger nonsynthetic DNA fragments. Multimers of transcription factor binding sites constructed by our method were used successfully in gel shift assays with homeodomain proteins (Ref. 4) and bZIP factors (unpublished results).
References 1 2 3 4
Hartley, J.L. and Gregori, T.J. (1981) Gene 13, 347–353. Liaw, G-J. (1994) BioTechniques 17, 668–670. Marsh, J.L., Erfle, M. and Wykes, E.J. (1984) Gene 32, 481–485 Meijer, A. H., et al. Plant J. in press.
Corresponding author: [email protected]
78
www.sciencedirect.com
c 1366-2120 1997 Elsevier Ltd. All rights reserved. PII: S1366-2120(08)70041-2
Technical Tips
Technical Tips Online, Vol. 2, 1997
FIGURE 1. A simple method for directional multimerization. Only the fragment and relevant restriction sites are shown, the rest of the vector is indicated with broken lines. Abbreviations: P, PstI; B, BamHI; Bg, BglII.
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A simple method for directional multimerization of DNA sequences Pieter B.F. Ouwerkerk and Johan Memelink Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, PO Box 9505, 2300 RA Leiden, The Netherlands
Keywords: Cloning & sequencing ▼We describe a simple method for the multimerization of DNA sequences in a directional manner. Multimers have a number of applications, especially in gene promoter analysis and for cloning of DNA-binding proteins using southwestern screening or the yeast one-hybrid system. Multimerization of a DNA sequence is usually performed by concatenation of synthetic oligonucleotides and subsequent cloning. Some strategies for directional multimerization have been described (Ref. 1, 2). However, we found that the considerable number of synthesis errors in many oligonucleotide preparations makes the selection of the correct multimer a laborious task. In addition, the yield of longer multimers in concatenation reactions is low, making the cloning of large multimers technically troublesome. Our method takes advantage of the polylinker sequences of the pIC vector series (Ref. 3). The DNA sequence of interest is cloned such that it is flanked by enzyme sites that generate compatible ends, which upon ligation are not recognized by either enzyme. The pIC vectors offer the compatible sticky-end combinations BamHI/BglII and SalI/XhoI. The example described here shows dimerization using BamHI/BglII (Fig. 1). The monomer fragment is excised with PstI/BglII and reintroduced between the PstI and BamHI sites of the monomer-containing plasmid. The resulting plasmid contains a head-to-tail dimer. Repeating the procedure with the dimer-containing plasmid results in a tetramer, and so on. Because multimerization proceeds
exponentially according to the formula 2n where n is the number of cloning steps starting with the monomer, large multimers are obtained after relatively few cloning steps. The monomer sequences are separated by the 6-bp fusions of the compatible restriction enzyme sites. In addition to BamHI/BgIII and SalI/XhoI fragments, any fragment can be cloned blunt-ended using the EcoRV and SmaI sites between the BamHI and BglII sites in the pIC-19H and 20H plasmids. In this case, multimerization results in monomers separated by 12 bp instead of the 6-bp BamHI/BglII or SalI/XhoI fusions. Besides its simplicity, a clear advantage of our method is that it starts with a monomer the sequence of which can be verified prior to the multimerization procedure. The method is not only applicable to short synthetic oligonucleotides, but also to larger nonsynthetic DNA fragments. Multimers of transcription factor binding sites constructed by our method were used successfully in gel shift assays with homeodomain proteins (Ref. 4) and bZIP factors (unpublished results).
References 1 2 3 4
Hartley, J.L. and Gregori, T.J. (1981) Gene 13, 347–353. Liaw, G-J. (1994) BioTechniques 17, 668–670. Marsh, J.L., Erfle, M. and Wykes, E.J. (1984) Gene 32, 481–485 Meijer, A. H., et al. Plant J. in press.
Corresponding author: [email protected]
78
www.sciencedirect.com
c 1366-2120 1997 Elsevier Ltd. All rights reserved. PII: S1366-2120(08)70041-2
Technical Tips
Technical Tips Online, Vol. 2, 1997
FIGURE 1. A simple method for directional multimerization. Only the fragment and relevant restriction sites are shown, the rest of the vector is indicated with broken lines. Abbreviations: P, PstI; B, BamHI; Bg, BglII.
www.sciencedirect.com
79