Optimization of chromatin immunoprecipitation
MetadataShow full item record
Transcription factors play a vital role in controlling cell growth, and locations of their binding sites at various points of the cell cycle can provide clues about malfunctions in eukaryotic growth, such as cancer. Our research focuses on transcriptional regulation of the eukaryotic cell cycle, using Saccharomyces cerevisiae as the model organism. We are observing binding patterns of affinity-tagged MCM1, SWI4, SWI5, FKH1, FKH2, and ACE2 transcription factors. These binding sites are discovered and isolated in vivo using chromatin immunoprecipitation (ChIP) followed by high-throughput, next-generation sequencing to map them to the genome for further analysis. The procedure locks transcription factors to their binding sites on DNA, and then eliminates the extraneous DNA to isolate the genes of binding site alone. However, ChIP generates a relatively low yield of DNA, often contaminated, and our research focuses on optimizing elements of the protocol to produce a higher, purer, output. Optimized lysis methods have reduced time and increased output of DNA, and sonication cycles have been adjusted to yield a more uniformly sheared mixture of DNA. The efficacy of sonication is evaluated through diagnostic gel electrophoresis and interpretation of visual results. In addition, the impact of pre-clearing on clarity of final yields was studied. DNA output and purity is repeatedly tested at various breakpoints of the ChIP procedure to ensure that the optimization modifications are delivering higher yields. DNA purity is tested using polymerase chain reaction (PCR) to find previously characterized target regions in the genome along with positive and negative controls to ensure homogeneity of the sample. Thus, by optimizing ChIP, we can obtain a highly accurate DNA sample more suitable for next-generation sequencing.