Protein--DNA Binding Assays and ChIP/Real-Time PCR for Transcript Abundance

EMSA principle

Protein-bound DNA migrates slower than free DNA, producing a shifted band

Filter binding quantification

increasing protein concentrations yield a binding curve from retained labeled DNA

DNA footprinting readout

Protected regions appear as missing bands after nuclease digestion of protein-bound DNA

ChiP workflow

Crosslink in vivo, fragment DNA, immunoprecipitate protein, then identify associated DNA (often by sequencing)

ChiP-seq capability

Genome-wide mapping of binding sites using high-throughout sequencing

Real Time PCR Quantification goal

qPCR estimates the starting amount of a specific transcript by fluorescence accumulation over cycles

Real-Time PCR Probe-and-quencher logic

A fluorescent reporter is quenched until polymerase extension displace/degrades the probe, increasing signal

Real-Time PCR Signal Interpretation

Higher starting template yields earlier and stronger fluorescence growth

Junction-spanning qPCR

primers/probes across exon-exon junctions selectively quantify specific splice isoforms

RT-PCR gel sizing

Different splice variants produce different amplicon sizes detectable by electrophoresis

RNA-seq profiling

Deep sequencing of cDNA quantifies many splice variants simultaneously