3C-digital PCR for quantification of chromatin interactions

BMC Molecular Biology, Dec 2016

Background 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 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. Results To 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). Conclusions Compared 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.

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3C-digital PCR for quantification of chromatin interactions

Du and Wang BMC Molecular Biol 3C-digital PCR for quantification of chromatin interactions Meijun Du 0 Liang Wang 0 0 Department of Pathology and MCW Cancer Center, Medical College of Wisconsin , Milwaukee, WI 53226 , USA Background: 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 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. Results: To 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 0.401.10) 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). Conclusions: Compared 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. Chromatin interaction; Chromosome conformation capture; Digital PCR; Quantitative PCR - Background Chromosome conformation capture (3C) has been widely used for detecting the physical interactions of chromosomal regions in  vivo [1]. In general, 3C library is first built by three basic steps involving fixation of chromatin spatial configuration by formaldehyde, digestion of cross-linked chromatin with restriction enzymes, and intra-molecular ligation of digested fragments that favors proximity. Chromatin interactions are then detected by measuring ligation frequency of two interacting fragments by polymerase chain reaction (PCR) [2–4]. Initial 3C assays estimate ligation frequency based on intensity of ethidium bromide-stained PCR products separated by agarose gel electrophoresis [3]. The gel-based assays, however, are hardly quantitative, making it difficult to differentiate subtle difference or detect weak signals. 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 © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. usually detects amplification signals at high cycle threshold (Ct) (such as Ct  ≥  35), which significantly reduces the assay’s sensitivity. In addition, current 3C-qPCR is complex because randomly ligated control is needed to normalize the amplification efficiency of different primer pairs. Recently, digital PCR (dPCR) has been emerged as a powerful tool for nucleic acid quantification, in particular, for rare molecule detection [6]. The technology detects number of targeted nucleic acids for absolute quantification by molecular counting. During dPCR, DNA samples are partitioned into thousands or millions of individual PCR reactions. Due to significant dilution, each reaction partition contains zero or (...truncated)


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Meijun Du, Liang Wang. 3C-digital PCR for quantification of chromatin interactions, BMC Molecular Biology, 2016, pp. 23, 17, DOI: 10.1186/s12867-016-0076-6