'Calling Cards' method for high-throughput identification of targets of yeast DNA-binding proteins

Haoyi Wang, Michael E. Heinz, Seth D. Crosby, Mark Johnston, Robi D. Mitra

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

We present a protocol for a novel method for identifying the targets of DNA-binding proteins in the genome of the yeast Saccharomyces cerevisiae. This is accomplished by engineering a DNA-binding protein so that it leaves behind in the genome a permanent mark - a 'calling card' - that provides a record of that protein's visit to that region of the genome. The calling card is the yeast Ty5 retrotransposon, whose integrase interacts with the Sir4 protein. If Sir4 is fused to a DNA-binding protein, it recruits the Ty5 integrase, which directs insertion of a Ty5 calling card into the genome. The calling card along with the flanking genomic DNA is harvested by inverse PCR and its genomic location is determined by hybridization of the product to a DNA microarray. This method provides a straightforward alternative to the 'ChIP-chip' method for determining the targets of DNA-binding proteins. This protocol takes ∼2 weeks to complete.

Original languageEnglish
Pages (from-to)1569-1577
Number of pages9
JournalNature Protocols
Volume3
Issue number10
DOIs
StatePublished - 2008

Fingerprint

Dive into the research topics of ''Calling Cards' method for high-throughput identification of targets of yeast DNA-binding proteins'. Together they form a unique fingerprint.

Cite this