A rapid simple approach to quantify chromosome conformation capture

Chromosome conformation capture (3C) is a power- ful tool to study DNA looping. The procedure gener- ates 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) deter- mined using SYBR Green, an inexpensive alterna- tive, is hampered by non-specific products and/or background fluorescence, both due to high tem- plate/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 tem- plate concentrations across different samples and apply it to 3C to assess looping among remote ele- ments identified by STAT1 and IRF1 ChIP-chip at the interferon-c responsive CIITA and SOCS1 loci. This rapid, inexpensive approach provided highly repro- ducible 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 asso- ciated with other approaches. Our data suggest that basal and induced looping is a involving remote enhancers is a common mechanism at IFNc-regulated targets.