Target Detection Assay (TDA): a versatile procedure to determine DNA blinding sites as demonstrated on SP1 protein (original) (raw)
Journal Article
Basel Institute for Immunology, Grenzacherstraβe 487
CH-4005 Basel, Switzerland
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Basel Institute for Immunology, Grenzacherstraβe 487
CH-4005 Basel, Switzerland
Search for other works by this author on:
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Hans-jürgen Thiesen, Christian Bach, Target Detection Assay (TDA): a versatile procedure to determine DNA blinding sites as demonstrated on SP1 protein, Nucleic Acids Research, Volume 18, Issue 11, 11 June 1990, Pages 3203–3209, https://doi.org/10.1093/nar/18.11.3203
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Abstract
We developed a rapid method designated TargetDetection Assay (TDA) to determine DNA binding sites for putative DNA binding proteins. A purified, functionally active DNA binding protein and a pool of random double-stranded oligonucleotides harbouring PCR primr sites at each end are included the TDA cycle which consists of four separate steps: a DNA protein incubation step, a protein DNA complex separation step , a DNA elutfon step and a polymerase chain reaction (PCR) DNA amplification step. The stringency of selection can be increased in consecutive TDA cycles. Since tiny amounts of retained DNA can be rescued by PCR, buffer systems, salt concentrations and competitor DNA contents can be varied In order to determine high affinity binding sites for the protein of choice. To test the efficiency of the TDA procedure potential DNA binding sites were selected by the DNA binding protein SP1 from a pool of oligonucleotides with random nucleotides at 12 positions. Target sites selected by recomblnant SP1 closely matched the SP1 consensus site. If DNA recognition sites have to be determined for known, mutated or putative DNA binding proteins, the Target Detection Assay (TDA) is a versatile and rapid technique for consideration.
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© 1990 Oxford University Press
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