Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP)

Mahmoud W. Yaish, Mingsheng Peng, Steven J. Rothstein

Research output: Chapter in Book/Report/Conference proceedingChapter

26 Citations (Scopus)

Abstract

DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages285-298
Number of pages14
Volume1062
ISBN (Print)9781627035798
DOIs
Publication statusPublished - 2014

Publication series

NameMethods in Molecular Biology
Volume1062
ISSN (Print)10643745

Fingerprint

DNA Methylation
Digestion
Methylation
pyrimidin-2-one beta-ribofuranoside
DNA
Genetic Epigenesis
Genes
Cytidine
Endonucleases
Enzymes
Eukaryota
Arabidopsis
Organism Cloning
Fishes
Databases
Polymerase Chain Reaction

Keywords

  • 5-azaC and zebularine
  • DNA methylation
  • MSAP
  • Mutant lines

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Yaish, M. W., Peng, M., & Rothstein, S. J. (2014). Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP). In Methods in Molecular Biology (Vol. 1062, pp. 285-298). (Methods in Molecular Biology; Vol. 1062). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-580-4_16

Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP). / Yaish, Mahmoud W.; Peng, Mingsheng; Rothstein, Steven J.

Methods in Molecular Biology. Vol. 1062 Humana Press Inc., 2014. p. 285-298 (Methods in Molecular Biology; Vol. 1062).

Research output: Chapter in Book/Report/Conference proceedingChapter

Yaish, MW, Peng, M & Rothstein, SJ 2014, Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP). in Methods in Molecular Biology. vol. 1062, Methods in Molecular Biology, vol. 1062, Humana Press Inc., pp. 285-298. https://doi.org/10.1007/978-1-62703-580-4_16
Yaish MW, Peng M, Rothstein SJ. Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP). In Methods in Molecular Biology. Vol. 1062. Humana Press Inc. 2014. p. 285-298. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-62703-580-4_16
Yaish, Mahmoud W. ; Peng, Mingsheng ; Rothstein, Steven J. / Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP). Methods in Molecular Biology. Vol. 1062 Humana Press Inc., 2014. pp. 285-298 (Methods in Molecular Biology).
@inbook{6c0b2009c2d544ba81601cdcc5cd08a4,
title = "Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP)",
abstract = "DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.",
keywords = "5-azaC and zebularine, DNA methylation, MSAP, Mutant lines",
author = "Yaish, {Mahmoud W.} and Mingsheng Peng and Rothstein, {Steven J.}",
year = "2014",
doi = "10.1007/978-1-62703-580-4_16",
language = "English",
isbn = "9781627035798",
volume = "1062",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "285--298",
booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP)

AU - Yaish, Mahmoud W.

AU - Peng, Mingsheng

AU - Rothstein, Steven J.

PY - 2014

Y1 - 2014

N2 - DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.

AB - DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic DNA digestion.

KW - 5-azaC and zebularine

KW - DNA methylation

KW - MSAP

KW - Mutant lines

UR - http://www.scopus.com/inward/record.url?scp=84934436860&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84934436860&partnerID=8YFLogxK

U2 - 10.1007/978-1-62703-580-4_16

DO - 10.1007/978-1-62703-580-4_16

M3 - Chapter

SN - 9781627035798

VL - 1062

T3 - Methods in Molecular Biology

SP - 285

EP - 298

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -