Comprehensive High‐Throughput Arrays for Relative Methylation (CHARM)

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

DNA methylation (DNAm) is a term used to describe the heritable covalent addition of a methyl group to cytosines at CpG dinucleotides in mammals. While methods for examining DNAm status at specific loci have existed for several years, recent technological advances have begun to enable the examination of DNAm across the genome. In this unit, we describe comprehensive high?throughput arrays for relative methylation (CHARM), a highly sensitive and specific approach to measure DNA methylation across the genome. This method makes no assumptions about where functionally important DNAm occurs, i.e., CpG island or promoter regions, and includes lower?CpG?density regions of the genome. In addition, it uses a novel genome?weighted smoothing algorithm to correct for CpG density and fragment biases present in methyl?enrichment or methyl?depletion DNA?fractionation methods. It can be applied to studying epigenomic changes in DNAm for normal and diseased samples. Curr. Protoc. Hum. Genet. 65:20.0.1?20.0.19. © 2010 by John Wiley & Sons, Inc.

Keywords: DNA methylation; CHARM; epigenome; methylome

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Introduction
Basic Protocol 1: CHARM Array Hybridization and Analysis
Support Protocol 1: Fractionation of Genomic DNA by Random Shearing
Support Protocol 2: Methyl‐Dependent Fractionation of Genomic DNA Using McrBC
Support Protocol 3: Whole‐Genome Amplification
Support Protocol 4: Determining McrBC Specificity and Unmethylated DNA Enrichment
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: CHARM Array Hybridization and Analysis

Materials

NimbleGen Array User's Guide downloaded from the Roche NimbleGen Web site (http://www.nimblegen.com/products/lit/methylation_userguide_v6p0.pdf)
NimbleScan Software User's Guide downloaded from the Roche NimbleGen Web site (http://www.nimblegen.com/products/lit/NimbleScan_v2p5_UsersGuide.pdf)
Bioconductor software (http://www.bioconductor.org), with the packages:
- oligo (http://bioconductor.org/packages/bioc/html/oligo.html)
- qvalue (http://bioconductor.org/packages/bioc/html/qvalue.html)
CHARM microarray annotation package: pd.feinberg.hg18.me.hx1 (http://rafalab.jhsph.edu/software.html)
NimbleScan feature extraction software (Roche NimbleGen)
3.5 µg untreated DNA sample (obtained using Support Protocols protocol 21 , protocol 32 , and protocol 43 ), analyzed by quantitative real‐time PCR to evaluate the specificity of McrBC in the digestion reaction ( protocol 5 )
3.5 µg methyl‐depleted DNA sample (obtained using Support Protocols protocol 21 , protocol 32 , and protocol 43 ), analyzed by quantitative real‐time PCR to evaluate the specificity of McrBC in the digestion reaction ( protocol 5 )
CHARM HD2 microarrays and supplies for HD2 array hybridization and processing (NimbleGen): see the NimbleGen Array User's Guide at the URL above
R Software (http://www.r‐project.org)

Support Protocol 1: Fractionation of Genomic DNA by Random Shearing

Materials

5 µg of high‐quality, high‐molecular‐weight, genomic DNA in 1× TE buffer ( appendix 3B )
1× TE buffer, pH 7.4 (Quality Biological), filtered prior to use to avoid clogging of the shearing assembly
0.2 M hydrochloric acid (wash solution I), filtered prior to use to avoid clogging of the shearing assembly
0.2 M sodium hydroxide (wash solution II), filtered prior to use to avoid clogging of the shearing assembly

50‐ml conical tubes (BD Falcon)
HydroShear device, equipped with a standard shearing assembly and syringe (DigiLab; http://www.digilabglobal.com/)

Support Protocol 2: Methyl‐Dependent Fractionation of Genomic DNA Using McrBC

Materials

Two 50‐µl aliquots of sheared genomic DNA: one labeled UT and the other labeled MD, ( protocol 2 )
10× NEBuffer2 (NEB2; New England Biolabs)
100 µg/ml (100×) bovine serum albumin (100× BSA)
100 mM (100×) guanosine triphosphate (GTP; store in single‐use aliquots at −80°C)
10 U/µL McrBC (New England Biolabs): test each new lot on a standard gDNA sample, using the digestion reaction and conditions provided below, to ensure complete digestion prior to use on any experimental samples
50% (w/v) glycerol
50 mg/ml proteinase K
SeaPlaque GTG Agarose (low melting point)
1× Tris‐acetate‐EDTA (TAE) buffer (see appendix 2D for 50×)
10 mg/ml ethidium bromide stock ( appendix 2D )
1 Kb Plus DNA Ladder (Invitrogen)
PhiDye (see recipe )
Qiagen Gel Extraction Kit including:
- Isopropanol
- Buffer QG
- Buffer PE
- Buffer EB

50° and 65°C water bath
500‐ml Erlenmeyer flask
Microwave oven
Clean 12‐in. × 14‐in. gel box with casting tray (USA Scientific)
Clean 8‐well, 1.5‐mm wide‐tooth combs for the 12‐in. × 14‐in. gel box (USA Scientific)
Kimwipes
Heat‐protective glove (“Hotglove”)
Gel imager (e.g., AlphaImager HP; Alpha Innotech, http://www.alphainnotech.com)
Razor blades

Additional reagents and equipment for ethanol precipitation of DNA ( appendix 3C ; use ammonium acetate as the monovalent cation) and agarose gel electrophoresis (see Ordway et al., , and unit 2.7 in this manual)

Support Protocol 3: Whole‐Genome Amplification

Materials

30 ng of excised, purified DNA from step 17 of protocol 3 for both MD and UT fractions
GenomePlex Complete Whole Genome Amplification (WGA2) kit (Sigma) including:
- 10× fragmentation buffer
- 1× library preparation buffer
- Library stabilization solution
- Library preparation enzyme
- 10× amplification master mix
- WGA DNA polymerase
- Nuclease‐free water
QiaQuick PCR Purification Kit including:
- Buffer PB
- Buffer PE
- Buffer EB

Additional reagents and equipment for spectrophotometrically quantitating DNA ( appendix 3D )

Support Protocol 4: Determining McrBC Specificity and Unmethylated DNA Enrichment

Materials

For each sample, 60 ng of untreated DNA (UT fraction) obtained after whole‐genome amplification ( protocol 4 ), at a concentration of 5 ng/µl
For each sample, 60 ng of McrBC‐treated DNA (MD fraction) obtained after whole‐genome amplification ( protocol 4 ), at a concentration of 5 ng/µl
Fast SYBR Green Master Mix (Applied Biosystems)
Quantitative real‐time PCR primers for the human genome (Ordway et al., ):
- GAPDH forward primer: TCTTGAGGCCTGAGCTACGTG
- GAPDH reverse primer: CCCGTCCTTGACTCCCTAGTGT
- GAPDH target sequence (5′ to 3′): CCTGCTGCCCACAGTCCAGTCCTGGG‐ AACCAGCACCGATCACCTCCCATCGGGCCAATCTCAGTCCCTT‐ CCCCCCTACGTCGGGGCCCACACGCTCGGTGCGTGCCCAGTTGAA‐ CCAGGCGGCTGCGGAAAAAAAAAAGCGGGGAGAAAGTAGGG‐ CCCGGCTACTAGCGGTTTTACGGGCG
- HIST1H2BA forward primer: AGTGCTGTGTAACCCTGGAAAA
- HIST1H2BA reverse primer: ACTCTCCTTACGGGTCCTCTTG
- HIST1H2BA target sequence: AGTGCTGTGTAACCCTGGAAAAGAACCGT‐ GTAACGCTGCAGAAGTGTGTGGTAGCTATGCCGGAGGTGTCAT‐ CTAAAGGTGCTACCATTTCCAAGAAGGGCTTTAAGAAAGCTGTCG‐ TTAAGACCCAGAAAAAGGAAGGCAAAAAGCGCAAGAGGACCCGTAAGGAGAGT
Milli‐Q water

384‐ or 96‐well optical plates
Optical plate seals
ABI 7900 HT real‐time PCR machine

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Figures

Figure 20.1.1 Overview of McrBC‐based fractionation (Lippman et al., ; Ordway et al., ) coupled with CHARM analysis (Irizarry et al., ). Genomic DNA is sheared to 1.5 to 3.0 kb and divided into two equal parts. The first is digested with McrBC, a methyl‐cytosine insensitive enzyme that recognizes Pu^m C(N_40‐3000 )^m CPu, and the second is untreated. Both fractions are then resolved side by side on a 1% agarose gel, and fragments between 1.65 kb and 3.0 kb are excised and purified. Next, the untreated fraction, representing total input DNA, is labeled with cyanine‐3 (Cy3) and the McrBC‐treated fraction, representing unmethylated DNA, is labeled with cyanine‐5 (Cy5) followed by cohybridization to a CHARM microarray. Sequences that are methylated will be present in the input fraction (Cy3) and depleted in the methyl‐depleted fraction (Cy5). For each probe on the array, a log ratio of the Cy3 to Cy5 intensity is calculated and represents the methylation level ( M ‐value) at each locus, with larger M ‐values representing more methylation and smaller M ‐values representing less methylation.

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Figure 20.1.2 Tab‐delimited sample description file (example).

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Figure 20.1.3 Table listing DMRs (example of output generated using commands in step 9).

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Figure 20.1.4 The expected size distribution, from 1.5 kb to 3.0 kb, of genomic DNA after shearing with a HydroShear device. We ran 500 ng of DNA on a 1% agarose gel in 1× TAE buffer for 60 min at 110 volts. Lane 1 contains a 1 Kb Plus DNA Ladder with the upper and lower arrows denoting 3.0 kb and 1.65 kb, respectively. Lane 2 contains unsheared, high‐quality, high‐molecular‐weight genomic DNA as a reference. Lane 3 contains genomic DNA that was sheared using a standard shearing assembly on a HydroShear device. The two straight horizontal lines denote the expected size distribution of DNA.

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Figure 20.1.5 Genomic DNA is size fractionated using a high‐molecular‐weight agarose gel. Mock (UT) and McrBC (MD) digestions were run side by side for 3.5 hr at 30 V. Lanes 1, 4, 5, 8, 9, 12, 13, and 16 contain a 1 Kb Plus DNA Ladder. Lanes 2 and 3 contain sheared gDNA from sample 1 that was mock digested (UT) and McrBC‐treated (MD), respectively. Lanes 6 and 7 contain sheared gDNA from sample 2 that was mock digested (UT) and McrBC‐treated (MD), respectively. Lanes 10 and 11 contain sheared gDNA from sample 3 that was mock digested (UT) and McrBC‐treated (MD), respectively. Lanes 14 and 15 contain sheared gDNA from sample 4 that was mock digested (UT) and McrBC‐treated (MD), respectively. Images on the left represent the agarose gel prior to extracting DNA and the images on the right show the agarose gel image after extracting DNA ranging in size from 1.65 to 3.0 kb. Well 1, 1 Kb Plus DNA Ladder (1 µg); Well 2, Sample 1–UT; Well 3, Sample 1–MD; Well 4, 1 Kb Plus DNA Ladder (1 µg); Well 5, 1 Kb Plus DNA Ladder (1 µg); Well 6, Sample 2–UT; Well 7, Sample 2–MD; Well 8, 1 Kb Plus DNA Ladder (1 µg); Well 9, 1 Kb Plus DNA Ladder (1 µg); Well 10, Sample 3–UT; Well 11, Sample 3–MD; Well 12, 1 Kb Plus DNA Ladder (1 µg); Well 13, 1 Kb Plus DNA Ladder (1 µg); Well 14, Sample 4–UT; Well 15, Sample 4–MD; Well 16, 1 Kb Plus DNA Ladder (1 µg).

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Figure 20.1.6 Suggested timeline for CHARM analysis of DNA methylation.

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Videos

Literature Cited

Literature Cited
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Comprehensive High‐Throughput Arrays for Relative Methylation (CHARM)

Abstract

Table of Contents

Materials

Basic Protocol 1: CHARM Array Hybridization and Analysis

Support Protocol 1: Fractionation of Genomic DNA by Random Shearing

Support Protocol 2: Methyl‐Dependent Fractionation of Genomic DNA Using McrBC

Support Protocol 3: Whole‐Genome Amplification

Support Protocol 4: Determining McrBC Specificity and Unmethylated DNA Enrichment

Figures

Videos

Literature Cited