A rapid simple approach to quantify chromosome conformation capture.
Chromosome conformation capture (3C) is a powerful tool to study DNA looping. The procedure generates chimeric DNA templates after ligation of restriction enzyme fragments juxtaposed in vivo by looping. These unique ligation products (ULPs) are typically quantified by gel-based methods, which are practically inefficient. Taqman probes may be used, but are expensive. Cycle threshold (Ct) determined using SYBR Green, an inexpensive alternative, is hampered by non-specific products and/or background fluorescence, both due to high template/ULP ratio. SYBR Green melting curve analysis (MCA) is a well-known qualitative tool for assessing PCR specificity. Here we present for the first time MCA as a quantitative tool (qMCA) to compare template concentrations across different samples and apply it to 3C to assess looping among remote elements identified by STAT1 and IRF1 ChIP-chip at the interferon-gamma responsive CIITA and SOCS1 loci. This rapid, inexpensive approach provided highly reproducible identification and quantification of ULPs over a significant linear range. Therefore, qMCA is a robust method to assess chromatin looping in vivo, and overcomes several drawbacks associated with other approaches. Our data suggest that basal and induced looping is a involving remote enhancers is a common mechanism at IFNgamma-regulated targets.
Abou El Hassan
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Genetics and Development Division, University Health Network, Department of Ophthalmology and Vision Sciences, University of Toronto, Toronto, Ontario, Canada M5T 2S8.
Bremner
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D045744:Cell Line, Tumor; D002875:Chromosomes / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004262:DNA Restriction Enzymes; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics
D005456:Fluorescent Dyes; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D015534:Trans-Activators; D004247:DNA; D004262:DNA Restriction Enzymes