Relative Efficiency of Transcription Factor Binding to Allelic Variants of Regulatory Regions of Human Genes in Immunoprecipitation and Real-Time PCR

Resumo

The efficiency at which specific transcription factors interact with DNA may vary in the presence of single nucleotide polymorphisms (SNPs), and the variation provides an important mechanism that regulates expression of human genes and contributes to the individual susceptibility to various diseases. Ample genetic and epigenetic data make it possible to predict both functional polymorphic variants and the transcription factors whose binding they affect. However, predictions of the kind require experimental verification. An original method developed for the purpose includes immunoprecipitation of DNA–protein complexes, followed by quantification of the bound DNA by real-time PCR. The method does not require chemical modification of the DNA probes and yield reproducible results with total nuclear extracts from cultured human cells.