Abstract
Tandemly repeated DNA sequences generated from single synthetic oligonucleotide monomers are useful for many purposes. With conventional ligation procedures low yields and random orientation of oligomers makes cloning of defined repeated sequences difficult. We solved these problems using 2 bp overhangs to direct orientation and random incorporation of linkers containing restriction sites during ligation. Ligation products are amplified by PCR using the linker oligonucleotides as primers. Restriction digestion of the PCR products generate multimer distributions whose length is controlled by the monomer/linker ratio. The concatenated DNA fragments of defined length, orientation and spacing can be directly used for subcloning or other applications without further treatment.
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Selected References
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- Dana S. L., Hoener P. A., Wheeler D. A., Lawrence C. B., McDonnell D. P. Novel estrogen response elements identified by genetic selection in yeast are differentially responsive to estrogens and antiestrogens in mammalian cells. Mol Endocrinol. 1994 Sep;8(9):1193–1207. doi: 10.1210/mend.8.9.7838152. [DOI] [PubMed] [Google Scholar]
- Jiang S. W., Eberhardt N. L. The human chorionic somatomammotropin gene enhancer is composed of multiple DNA elements that are homologous to several SV40 enhansons. J Biol Chem. 1994 Apr 8;269(14):10384–10392. [PubMed] [Google Scholar]
- Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maxwell I. H., Harrison G. S., Wood W. M., Maxwell F. A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription. Biotechniques. 1989 Mar;7(3):276–280. [PubMed] [Google Scholar]
- Schafer A. J., Fournier R. E. Multiple elements regulate phosphoenolpyruvate carboxykinase gene expression in hepatoma hybrid cells. Somat Cell Mol Genet. 1992 Nov;18(6):571–581. doi: 10.1007/BF01232653. [DOI] [PubMed] [Google Scholar]
- Veldman G. M., Lupton S., Kamen R. Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression. Mol Cell Biol. 1985 Apr;5(4):649–658. doi: 10.1128/mcb.5.4.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang W. W., Farrés J., Busch H. DNA footprint enhancement using tandem binding sites. Biotechniques. 1991 Dec;11(6):728, 730, 732-3. [PubMed] [Google Scholar]
