3C-digital PCR for quantification of chromatin interactions.

Meijun Du,Liang Wang

doi:10.1186/s12867-016-0076-6

Abstract

BackgroundChromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of 3C is to convert physical chromatin interactions into specific DNA ligation products, which are then detected by quantitative polymerase chain reaction (qPCR). However, 3C-qPCR assays are often complicated by the necessity of normalization controls to correct for amplification biases. In addition, qPCR is often limited to a certain cycle number, making it difficult to detect fragment ligations with low frequency. Recently, digital PCR (dPCR) technology has become available, which allows for highly sensitive nucleic acid quantification. Main advantage of dPCR is its high precision of absolute nucleic acid quantification without requirement of normalization controls.ResultsTo demonstrate the utility of dPCR in quantifying chromatin interactions, we examined two prostate cancer risk loci at 8q24 and 2p11.2 for their interaction target genes MYC and CAPG in LNCaP cell line. We designed anchor and testing primers at known regulatory element fragments and target gene regions, respectively. dPCR results showed that interaction frequency between the regulatory element and MYC gene promoter was 0.7 (95% CI 0.40–1.10) copies per 1000 genome copies while other regions showed relatively low ligation frequencies. The dPCR results also showed that the ligation frequencies between the regulatory element and two EcoRI fragments containing CAPG gene promoter were 1.9 copies (95% CI 1.41–2.47) and 1.3 copies per 1000 genome copies (95% CI 0.76–1.92), respectively, while the interaction signals were reduced on either side of the promoter region of CAPG gene. Additionally, we observed comparable results from 3C-dPCR and 3C-qPCR at 2p11.2 in another cell line (DU145).ConclusionsCompared to traditional 3C-qPCR, our results show that 3C-dPCR is much simpler and more sensitive to detect weak chromatin interactions. It may eliminate multiple and complex normalization controls and provide accurate calculation of proximity-based fragment ligation frequency. Therefore, we recommend 3C-dPCR as a preferred method for sensitive detection of low frequency chromatin interactions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-016-0076-6) contains supplementary material, which is available to authorized users.

Highlights

Chromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo
Our results show that chromosome conformation capture (3C)-digital PCR (dPCR) is easier to use and more sensitive in determining chromatin interactions
Because ligation frequency is generally low between any two non-adjacent fragments [1, 5], a meaningful 3C analysis critically relies on the accurate quantification of different ligation products

Summary

Introduction

Chromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of 3C is to convert physical chromatin interactions into specific DNA ligation products, which are detected by quantitative polymerase chain reaction (qPCR). QPCR is often limited to a certain cycle number, making it difficult to detect fragment ligations with low frequency. Chromatin interactions are detected by measuring ligation frequency of two interacting fragments by polymerase chain reaction (PCR) [2,3,4]. With advent of real-time quantitative PCR (qPCR), quantification of 3C ligation frequency becomes more accurate by monitoring the signal strength after each amplification cycle [4]. Due to relatively low ligation frequency in 3C library [1, 5], the qPCR assay

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