Chromosome Conformation Capture Carbon Copy Technology

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Chromosome conformation capture (3C) is used to quantify physical DNA contacts in vivo at high resolution. 3C was first used in yeast to map the spatial chromatin organization of chromosome III, and in higher eukaryotes to demonstrate that genomic DNA elements regulate target genes by physically interacting with them. 3C has been widely adopted for small?scale analysis of functional chromatin interactions along (cis ) or between (trans ) chromosomes. For larger?scale applications, chromosome conformation capture carbon copy (5C) combines 3C with ligation?mediated amplification (LMA) to simultaneously quantify hundreds of thousands of physical DNA contacts by microarray or ultra?high?throughput DNA sequencing. 5C allows the mapping of extensive networks of physical interactions among large sets of genomic elements throughout the genome. Such networks can provide important biological insights, e.g., by identifying relationships between regulatory elements and their target genes. This unit describes 5C for large?scale analysis of cis ? and trans ?chromatin interactions in mammalian cells. Curr. Protoc. Mol. Biol. 80:21.14.1?21.14.13. © 2007 by John Wiley & Sons, Inc.

Keywords: chromatin structure; epigenetic regulation; genomics; systems/network biology; DNA looping; transcription; high throughput

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Introduction
Strategic Planning
Basic Protocol 1: Generation of 5C Libraries from Cells and Control BAC DNA
Basic Protocol 2: Quality Control of 5C Libraries by Nested PCR
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Generation of 5C Libraries from Cells and Control BAC DNA

Materials

∼125 ng/µl cellular 3C template library in TE buffer (see unit 21.11 )
∼50 ng/µl control template library in TE buffer (see unit 21.11 )
9.9 mg/ml salmon testis DNA (Sigma)
Deionized water, autoclaved
20 µM 5C primers (see ) in 1× TE buffer ( appendix 22 )
10× 5C annealing buffer (see recipe )
10× 5C ligation buffer (see recipe )
40 U/µl Taq DNA ligase (NEB)
10× PCR buffer (see recipe )
50 mM MgSO₄
25 mM dNTP mix
80 µM forward T7 and reverse T3 universal PCR primer stock solutions in 1× TE buffer ( appendix 22 )
5 U/µl Taq DNA polymerase (NEB)
2% agarose gels containing 0.5× TBE and 0.5 µg/ml ethidium bromide (see unit 2.5 )
DNA molecular weight marker of known concentration (e.g., 100‐bp ladder; NEB)

0.5‐ml PCR tubes or 96‐well plates
Refrigerated tabletop centrifuge
Automated thermal cycler
MinElute PCR purification kit (Qiagen)
Gel documentation system with appropriate software for quantifying PCR products

Additional reagents and equipment for performing PCR (units 15.1 & 15.7 ) and agarose gel electrophoresis (unit 2.5 )

NOTE: It is important to read all protocol steps before starting the experiment.

Basic Protocol 2: Quality Control of 5C Libraries by Nested PCR

Materials

5C library ( protocol 1 )
10× PCR buffer (see recipe )
50 mM MgSO₄
25 mM dNTP mix
80 µM nested 5C primers (see ) in 1× TE buffer ( appendix 22 )
5 U/µl Taq DNA polymerase (NEB)
2% agarose gels containing 0.5× TBE and 0.5 µg/ml ethidium bromide (see unit 2.5 )
DNA molecular weight markers of known concentration (e.g., 100‐bp ladder; NEB)

96‐well plates
Automated thermal cycler
Gel documentation system with appropriate software for quantifying PCR products

Additional reagents and equipment for performing PCR (units 15.1 & 15.7 ) and agarose gel electrophoresis (unit 2.5 )

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Figures

Figure 21.14.1 Diagram of 3C (left) and 5C (right) methods. 3C generates a library of ligation products each of which reflects a physical interaction between the corresponding loci in the cell. 5C employs ligation‐mediated amplification to detect specific ligation products in the 3C library. 5C is performed at high levels of multiplexing with thousands of 5C primers to detect millions of ligation products. 5C products can be quantified by microarray analysis or high‐throughput sequencing.

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Figure 21.14.2 Design of 5C primers. Top: Forward and reverse primers are composed of specific parts that recognize the ends of specific restriction fragments, and universal tails that are used for PCR amplification of the 5C library. Middle: Forward and reverse primers anneal to opposite strands along the genome, but they will anneal to the same strand and directly across the restriction site of head‐to‐head ligation products in the 3C library. Bottom: Universal primers that recognize the tails of 5C primers are used to amplify the entire 5C library. Nested primers that recognize individual 5C primers are used to amplify and quantify specific 5C products.

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Figure 21.14.3 Example of a 5C titration. 5C was performed with increasing amounts of 3C control library and the resulting 5C products were amplified with universal primers. 5C products were analyzed by agarose gel electrophoresis (top) and a titration curve was generated from the quantitated band signals (bottom). No 5C ligation products were observed when either 3C template or Taq ligase was left out. Increasing amounts of 3C library yield increasing amounts of 5C products, indicating that 5C quantitatively detects 3C ligation products. When performing 5C, it is best to use an amount of 3C template that falls within the linear range of 5C product formation.

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Figure 21.14.4 A theoretical 5C experiment to detect long‐range interactions between a set of three promoters and all surrounding chromatin segments. Top: Reverse 5C primers are designed for the three promoters and forward 5C primers for all other chromatin segments throughout the region of interest. All 5C primers are pooled and used for a single 5C analysis to quantify all interactions between the three promoters and all surrounding segments in parallel. Middle: Theoretical outcome of the 5C experiment. Interaction frequencies are generally inversely related to the genomic distance between the corresponding chromatin segments. Bottom: Local peaks in interaction frequencies indicate specific long‐range interactions.

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Videos

Literature Cited

Literature Cited
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