
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.
Electrophoresis, Agar Gel, Nuclear Proteins, DNA, DNA Restriction Enzymes, Nucleic Acid Denaturation, Polymerase Chain Reaction, Chromosomes, Cell Line, Tumor, Trans-Activators, Methods Online, Humans, Nucleic Acid Conformation, Fluorescent Dyes, HeLa Cells
Electrophoresis, Agar Gel, Nuclear Proteins, DNA, DNA Restriction Enzymes, Nucleic Acid Denaturation, Polymerase Chain Reaction, Chromosomes, Cell Line, Tumor, Trans-Activators, Methods Online, Humans, Nucleic Acid Conformation, Fluorescent Dyes, HeLa Cells
| selected citations These citations are derived from selected sources. This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically). | 20 | |
| popularity This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network. | Top 10% | |
| influence This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically). | Top 10% | |
| impulse This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network. | Top 10% |
