Polymerase Chain Reaction (PCR) Detection of 3C Ligation Products Present in 3C, ChIP-Loop, and Control Libraries: Library Titration and Interaction Frequency Analysis

Abstract

In all 3C-based assays, the aim is to measure the frequency with which two loci interact within cells. This is achieved by determining the relative abundance of ligation products formed during the 3C protocol that carry sequences from both loci. Individual 3C ligation products that are present in a 3C library, the ChIP-loop library, or the control library can be detected by polymerase chain reaction (PCR) using locus-specific primers. The following protocol outlines the procedure to measure relative abundances of ligation products in these libraries using PCR. The first part of the protocol-library titration-is required only once to determine the linear range of PCR detection. Once the linear range of PCR detection is known for a given ligation product library, follow the second part-interaction analysis-to measure the abundance of ligation products of interest in that library. Specific issues regarding determining the relative abundance of ligation products, designing PCR primers, and quantifying interaction frequencies are also described.