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Methylation‐Specific PCR

Bradford Coffee¹

¹Emory University School of Medicine, Atlanta, Georgia

Publication Name:

Current Protocols in Human Genetics

Unit Number:

Unit 10.6

DOI:

10.1002/0471142905.hg1006s61

Online Posting Date:

April, 2009

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Abstract

DNA methylation is a dynamic covalent modification of DNA that is found in transcriptionally repressed regions of the genome. In concert with other epigenetic modifications, such as the repressive histone H3 modifications of lysine 9 and lysine 27 methylation, DNA methylation acts to repress gene expression through the formation of condensed chromatin structures. Thus, DNA methylation is a marker for gene repression. Methylation-specific PCR (MSP) is a rapid and inexpensive method that can be used to determine the methylation status of DNA. MSP can be used to assess CpG methylation at imprinted genes and on the inactive X chromosome. In addition, MSP can be used to investigate the aberrant DNA methylation that occurs in diseases such as fragile X syndrome and in many types of cancers. MSP is rapid, inexpensive, and relatively simple to perform and provides a powerful tool to investigate these epigenetic processes. Curr. Protoc. Hum. Genet. 61:10.6.1-10.6.14. © 2009 by John Wiley & Sons, Inc.

Keywords: DNA methylation; sodium bisulfite; real-time PCR; imprinting

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Introduction
Basic Protocol 1: Sodium Bisulfite Treatment of Genomic DNA
Basic Protocol 2: Methylation-Sensitive PCR
Basic Protocol 3: Quantitative Methylation-Sensitive PCR (Q-MSP)
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Sodium Bisulfite Treatment of Genomic DNA

Materials

<1 µg sample and control (from patients affected with Prader-Willi syndrome—with absence of unmethylated paternally inherited alleles, and Angelman syndrome—with absence of methylated maternally inherited alleles) DNA (for preparation see, e.g., appendix 3B)
Distilled water, room temperature and 65°C (autoclaved)
2 M and 3 M NaOH, freshly prepared (see recipe)
10 mM hydroquinone, freshly prepared (see recipe)
3.6 M sodium bisulfite, freshly prepared (see recipe)
Mineral oil
Wizard genomic DNA purification system (Promega), or equivalent, including:
- Collection tubes
- 0.5 M EDTA, pH 8.0
- Nuclease-free water
- Nuclei lysis solution
- RNase A solution
- Wizard SV lysis buffer
- Wizard SV minicolumns
- Wizard SV wash solution
95% (v/v) ethanol
10 mg/ml glycogen
3 M sodium acetate, pH 5.2
100% and 70% (v/v) ethanol, ice cold

1.5-ml microcentrifuge tube
54°C heating block

NOTE: All centrifugations, unless otherwise noted, are at maximum speed in a microcentrifuge (>20,000 × g).

NOTE: Before setting up the bisulfite reactions, it is important that all chemicals used in the reaction—sodium hydroxide, hydroquinone, and sodium bisulfite—be prepared fresh just before use. The amounts given can be scaled to produce the amount needed.

Basic Protocol 2: Methylation-Sensitive PCR

Materials

HotStar Taq DNA polymerase kit (Qiagen), including:
- 10× PCR buffer II, without MgCl₂
- 25 mM MgCl₂
- HotStar Taq DNA polymerase
10 mM dNTPs (appendix 2D)
PW/AS primer cocktail:
- 1 pmol/µl methylated DNA–specific MF1 primers (5¢-TAAATAAGTACGTTTGCGCGGTC-3¢)
- 1 pmol/µl methylated MR1 DNA–specific primers (5¢-AACCTTACCCGCTCCATCGCG-3¢)
- 2 pmol/µl unmethylated DNA–specific PF2 primers (5¢-GTAGGTTGGTGTGTATGTTTAGGT-3¢)
- 2 pmol/µl unmethylated DNA–specific PR2 primers (5¢-ACATCAAACATCTCCAACAACCA-3¢)
- 1 pmol/µl unmodified DNA–specific SNRPNF primers (5¢-GGAGGGAGCTGGGACCCC-3¢)
- 1 pmol/µl unmodified DNA–specific SNRPNR primers (5¢-GAAGCCACCGGCACAGCT-3¢)
Distilled water, autoclaved
Sodium bisulfite–treated sample and control DNA (Basic Protocol 1)
3% (w/v) agarose gel
100- to 300-bp molecular size markers (e.g., Promega 100-bp ladder)
Ethidium bromide

Thermal cycler and appropriate PCR tubes
UV transilluminator

Additional reagents and equipment for performing agarose gel electrophoresis (unit 2.7)

Basic Protocol 3: Quantitative Methylation-Sensitive PCR (Q-MSP)

Materials

Control DNA from a normal individual (for preparation see, e.g., appendix 3B)
Sodium bisulfite–treated standard DNA (Basic Protocol 1) from a cytogenetically identified duplication of the H19 region of 11p15 (if available)
Sodium bisulfite–treated sample and control DNA (Basic Protocol 1)
10× PCR buffer (Invitrogen)
50 mM MgCl₂ (Invitrogen)
1.25 mM dNTPs (appendix 2D)
Amplification primers
- CTCF6F (5¢-GTATAGTATATGGGTATTTTTGGAGG-3¢)
- CTCF6R (5¢-CCCAATTAAAACRAACTCRAACTATAAT-3¢)
Dual-labeled fluorescent probes with Black Hole Quencher 1 (BHQ1) added by supplier during synthesis
- CTCF6M: 5¢-FAM-AAGTGGTCGCGCGGCGGTAGTGTA-BHQ1-3¢ (IDT)
- CTCF6U: 5¢-HEX-TGGAAGTGGTTGTGTGGTGGTAGTGTAGG-BHQ1-3¢ (IDT)
5 U/µl Platinum Taq DNA polymerase (Invitrogen)
Distilled water (autoclaved)

8-well strip cap tube
iQ5 iCycler thermal cycler with iQ5 software (BioRad)

Additional reagents and equipment for preparing sodium bisulfite–treated DNA (Basic Protocol 1) and quantifying DNA by absorption spectroscopy (appendix 3D)

Looking for materials? Suppliers Guide

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Figures

Figure 10.6.1

Flow chart for methylation detection using bisulfite treatment and PCR. Bisulfite treatment converts unmethylated Cs to Us. Because PCR amplification then proceeds using a primer containing complementary As rather than Gs, the products of the bisulfite-treated unmethylated template will contain Ts in place of unmethylated Cs.

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Figure 10.6.2

Analysis of DNA methylation at the imprinted SNRPN promoter. Lane 1, negative; lane 2, Prader-Willi syndrome; lane 3, Angelman syndrome; lane 4, water; lane 5, unmodified DNA.

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Figure 10.6.3

Standard curve for H19 methylated and unmethylated DNA.

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Figure 10.6.4

Amplification plot from a Q-MSP assay.

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Literature Cited

Literature Cited
	Antequera, F., Boyes, J., and Bird, A. 1990. High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell 62:503-514.
	Coffee, B., Muralidharan, K., Highsmith, W.E. Jr., Lapunzina, P., and Warren, S.T. 2006. Molecular diagnosis of Beckwith-Wiedemann syndrome using quantitative methylation-sensitive polymerase chain reaction. Genet. Med. 8:628-634.
	Eads, C.A., Danenberg, K.D., Kawakami, K., Saltz, L.B., Blake, C., Shibata, D., Danenberg, P.V., and Laird, P.W. 2000 MethyLight: A high-throughput assay to measure DNA methylation. Nucleic Acids Res. 28:E32.
	Frommer, M., McDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L., and Paul, C.L. 1992. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. U.S.A. 89:1827-1831.
	Gonzalgo, M.L. and Jones, P.A. 1997. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 25:2529-2531.
	Graff, J.R., Herman, J.G., Lapidus, R.G., Chopra, H., Xu, R., Jarrard, D.F., Isaacs, W.B., Pitha, P.M., Davidson, N.E., and Baylin, S.B. 1995. E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas. Cancer Res. 55:5195-5199.
	Herman, J.G., Latif, F., Weng, Y., Lerman, M.I., Zbar, B., Liu, S., Samid, D., Duan, D.S., Gnarra, J.R., Linehan, W.M., and Baylin, S.B. 1994. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc. Natl. Acad. Sci. U.S.A. 91:9700-9704.
	Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D., and Baylin, S.B. 1996a. Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. U.S.A. 93:9821-9826.
	Herman, J.G., Jen, J., Merlo, A., and Baylin, S.B. 1996b. Hypermethylation-associated inactivation indicates a tumor suppressor role for p15(INK4B). Cancer Res. 56:722-727.
	Kubota, T., Das, S., Christian, S.L., Baylin, S.B., Herman, J.G., and Ledbetter, D.H. 1997. Methylation-specific PCR simplifies imprinting analysis. Nat. Genet. 16:16-17.
	Li, E., Beard, C., and Jaenisch, R. 1993. Role for DNA methylation in genomic imprinting. Nature 366:362-365.
	Merlo, A., Herman, J.G., Mao, L., Lee, O.J., Gabrielson, E., Burger, P.C., Baylin, S.B., and Sidransky, D. 1995. 5¢ CpG island methylation is associated with transcriptional silencing of the tumour suppressor P16/CDKN2/MTS1 in human cancers. Nat. Med. 1:686-692.
	Riggs, A.D. and Pfeifer, G.P. 1992. X-chromosome inactivation and cell memory. Trends Genet. 8:169-174.
	Xiong, Z. and Laird, P.W. 1997. COBRA: A sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 25:2532-2534.