Abstract
The propensity of Taq polymerase to add 3′-A overhangs (1,2) to PCR-amplified DNA has made possible a simple method for cloning PCR products into a T-vector (Invitrogen [San Diego, CA]; 3–5). Here we present a related strategy that uses T-linkers to add sequences, such as restriction sites, to the ends of PCR products (see Note 1). A single base T overhang at the end of a synthetic double-stranded oligonucleotide linker allows ligation of the linker to the unpolished ends of a PCR product. This avoids the expense of adding the “extra” sequences to sequence-specific primers.
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References
Clark, J. M. (1988) Novel nontemplated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16, 9677–9686.
Mole, S. E., Iggo, R. D., and Lane, D. P. (1989) Using the polymerase chain reaction to modify expression plasmids for epitope mapping. Nucleic Acids Res. 17, 3319.
Mead, D. A., Pey, N. K., Herrnstadt, C., Marcil, R. A., and Smith, L. M. (1991) A universal method for the direct cloning of PCR amplified nucleic acid. Bio/Technology 9, 657–663.
Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19, 1154.
Holton, T. A. and Graham, M. W. (1991) A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res. 19, 1156.
Hoppe, B. L., Conti-Tronconi, B. M., and Horton, R. H. (1992) Gel loading dyes compatible with PCR. BioTechniques 12, 679,680.
Horton, R. M., Hoppe, B. L., and Conti-Tronconi, B. M. (1994) AmpliGrease, “Hot Start” PCR using petroleum jelly. BioTechniques 16, 42,43.
Tabor, S. and Richardson, C. C. (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82, 1074–1078.
Ko, M. S. H., Takahashi, N., Nishiguchi, K., and Abe, K. (1990) Unbiased amplification of a highly complex mixture of DNA fragments by ‘lone-linker’-tagged PCR. Nucleic Acids Res. 18, 4293.
Bhat, G. J., Lodes, M. J., Myler, P. J., and Stuart, K. D. (1991) A simple method for cloning blunt ended DNA fragments. Nucleic Acids Res. 19, 398.
Meissner, P. S. Sisk, W. P., and Berman, M. L. (1987) Bacteriophage lambda cloning system for the construction of directional cDNA libraries. Proc. Natl. Acad. Sci. USA 84, 4171–4175.
Rich, J. J. and Willis, D. K. (1990) A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction. Nucleic Acids Res. 18, 6673–6676.
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© 1997 Humana Press Inc.
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Horton, R.M., Raju, R., Conti-Fine, B.M. (1997). A T-Linker Strategy for Modification and Directional Cloning of PCR Products. In: White, B.A. (eds) PCR Cloning Protocols. Methods in Molecular Biology™, vol 67. Humana Press, Totowa, NJ. https://doi.org/10.1385/0-89603-483-6:101
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DOI: https://doi.org/10.1385/0-89603-483-6:101
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-483-9
Online ISBN: 978-1-59259-553-2
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