Viggiani, C. J., Knott, S. R., Aparicio, O. M. (February 2010) Genome-wide analysis of DNA synthesis by BrdU immunoprecipitation on tiling microarrays (BrdU-IP-chip) in Saccharomyces cerevisiae. Cold Spring Harb Protoc, 2010 (2). ISSN 1559-6095
Abstract
The incorporation of thymidine analogs, such as 5-bromo-2′-deoxyuridine (BrdU), into newly synthesized DNA is a powerful tool for analysis of DNA replication, repair, and other aspects of DNA metabolism. In Saccharomyces cerevisiae, several assays have been developed to identify chromosomal DNA that has incorporated BrdU. Here we describe an approach that couples BrdU immunoprecipitation with DNA microarrays (BrdU-IP-chip) supplied by Roche NimbleGen to enable the genome-wide identification of BrdU-labeled chromosomal DNA. In this procedure, yeast cells that have been engineered to assimilate BrdU from the growth medium and incorporate it into replicating DNA are grown in the presence of BrdU under experimental conditions. These cells are harvested, the genomic DNA is isolated and randomly sheared, and the BrdU-labeled DNA is then immunoprecipitated. This immunoprecipitated DNA is polymerase chain reaction (PCR)-amplified, labeled with a fluorophore, and cohybridized along with a reference sample (typically total genomic DNA not subject to immunoprecipitation, but amplified and labeled with a different fluorophore) onto DNA microarrays. The data are then normalized, and chromosomal regions of BrdU incorporation (BrdU peaks) are identified. BrdU peak heights can be quantified and compared with BrdU peak heights at different genomic locations or under different experimental conditions (e.g., different mutants or time points). BrdU-IP-chip has many potential applications and has already been used to identify replication origins, make quantitative comparisons of origin firing between strains, and examine replication fork progression.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Bromodeoxyuridine/*chemistry DNA Replication Genes, Fungal *Genetic Techniques Immunoprecipitation/methods Models, Genetic Mutation Oligonucleotide Array Sequence Analysis/*methods Polymerase Chain Reaction Saccharomyces cerevisiae/*genetics Sequence Analysis, DNA/*methods |
| Subjects: | bioinformatics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification bioinformatics > genomics and proteomics > genetics & nucleic acid processing > genomes organism description > yeast |
| CSHL Authors: | |
| Communities: | CSHL Post Doctoral Fellows CSHL labs > Hannon lab |
| Depositing User: | Matt Covey |
| Date: | February 2010 |
| Date Deposited: | 18 Mar 2015 15:01 |
| Last Modified: | 18 Mar 2015 15:01 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/31277 |
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