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Nucleic Acids Research, 1991, Vol. 19, No. 7 1449-1453
© 1991

MOLECULAR BIOLOGY

Binding site selection analysis of protein-DNA interactions via solid phase sequencing of oligonucleotide mixtures

Joseph A. Gogos1, George Tzertzinis1 and Fotis C. Kafatos1,2,*

1Department of Cellular and Developmental Biology, The Biological Laboratories, Harvard University 16 Divinity Avenue, Cambridge, MA 02138, USA 2Institute of Molecular Biology and Biotechnology, Research Center of Crete PO Box 1527, Heraklion 71110, Crete, Greece

* To whom correspondence should be addressed at Harvard University

Received January 14, 1991. Revised March 1, 1991. Accepted March 1, 1991.

By combining the concept of degenerate oligonucleotide mutagenesis (1,2,3,4) and the convenience of solid phase chemical DNA sequencing (5), we have developed a rapid procedure for determining the specificity of DNA-binding proteins in vitro. Starting with a degenerate oligonucleotide mixture, the technique assays for alternative nucleotides in fractions that are bound or non-bound to the protein of interest. In contrast to previous approaches using degenerate oligonucleotides, it does not involve cloning but rather employs direct sequencing of the oligonucleotide mixtures after attachment to a solid support. Solid state processing obviates the need for both DNA extractions from polyacrylamide gels and time-consuming ethanol precipitations. Because of its convenience and sensitivity, this binding site selection analysis is well suited to determining rapidly the sequence preference of DNA-binding proteins that are available in small amounts, and complements well established approaches like methylation interference or missing contact assays. The solid phase reaction protocol we propose can also improve these latter approaches.


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J. Gogos, T Hsu, J Bolton, and F. Kafatos
Sequence discrimination by alternatively spliced isoforms of a DNA binding zinc finger domain
Science, September 25, 1992; 257(5078): 1951 - 1955.
[Abstract] [PDF]


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