CoChat
Sign Up
Cited by 1,033Epigenetics and DNA MethylationGenetic Syndromes and ImprintingGenomics and Chromatin Dynamics

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

Open access
Yang, A. S.·2004·Nucleic Acids Research
DOI 10.1093/nar/gnh032PMC PMC373427
Open this paper in CoChat — free account

Abstract

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15 000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1093/nar/gnh032

Cites

1 of 1 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Maternal PM2.5 exposure has been correlated with overall placental DNA mutation rate, evidenced by an increased Alu rate and altered DNA methylation in promoter regions of key DNA repair and tumor suppressor genes including APEX1, ERCC4, DAPK, and PARP1.

    “Additionally, maternal PM2.5 exposure has been correlated with overall placental DNA mutation rate, evidenced by an increased Alu rate (as an estimate of global methylation [188]) and altered DNA methylation in the promoter regions of the key DNA repair and tumor suppressor genes including APEX1, ERCC4, DAPK, and PARP1 [189, 190].”

    From Air pollution and children’s health—a review of adverse effects associated with prenatal exposure from fine to ultrafine particulate matter (2021) · cites this paper as [188]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 27

  1. [2]Jones P.A. and Takai,D. (2001) The role of DNA methylation in mammalian epigenetics. Science, 293, 1068–1070. [DOI] [PubMed] [Google Scholar]
  2. [3]Jones P.A. and Baylin,S.B. (2002) The fundamental role of epigenetic events in cancer. Nature Rev. Genet., 3, 415–428. [DOI] [PubMed] [Google Scholar]
  3. [4]Issa J.P. (2000) CpG-island methylation in aging and cancer. Curr. Top. Microbiol. Immunol., 249, 101–118. [DOI] [PubMed] [Google Scholar]
  4. [5]Richardson B. (2003) Impact of aging on DNA methylation. Ageing Res. Rev., 2, 245–261. [DOI] [PubMed] [Google Scholar]
  5. [6]Glover A.B., Leyland-Jones,B.R., Chun,H.G., Davies,B. and Hoth,D.F. (1987) Azacitidine: 10 years later. Cancer Treat. Rep., 71, 737–746. [PubMed] [Google Scholar]
  6. [7]Santini V., Kantarjian,H.M. and Issa,J.P. (2001) Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann. Intern. Med., 134, 573–586. [DOI] [PubMed] [Google Scholar]
  7. [8]Clark S.J., Harrison,J., Paul,C.L. and Frommer,M. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res., 22, 2990–2997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. [9]Xiong Z. and Laird,P.W. (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res., 25, 2532–2534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. [10]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. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. [11]Herman J.G., Graff,J.R., Myèohèanen,S., Nelkin,B.D. and Baylin,S.B. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl Acad. Sci. USA, 93, 9821–9826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. [12]Uhlmann K., Brinckmann,A., Toliat,M.R., Ritter,H. and Nurnberg,P. (2002) Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis. Electrophoresis, 23, 4072–4079. [DOI] [PubMed] [Google Scholar]
  12. [13]Wagner I. and Capesius,I. (1981) Determination of 5-methylcytosine from plant DNA by high-performance liquid chromatography. Biochim. Biophys Acta, 654, 52–56. [DOI] [PubMed] [Google Scholar]
  13. [14]Feinberg A.P. and Vogelstein,B. (1983) Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature, 301, 89–92. [DOI] [PubMed] [Google Scholar]
  14. [15]Bestor T.H., Hellewell,S.B. and Ingram,V.M. (1984) Differentiation of two mouse cell lines is associated with hypomethylation of their genomes. Mol. Cell. Biol., 4, 1800–1806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. [16]Friso S., Choi,S.W., Girelli,D., Mason,J.B., Dolnikowski,G.G., Bagley,P.J., Olivieri,O., Jacques,P.F., Rosenberg,I.H., Corrocher,R. et al. (2002) A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status. Proc. Natl Acad. Sci. USA, 99, 5606–5611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. [17]Antequera F., Tamame,M., Villanueva,J.R. and Santos,T. (1984) DNA methylation in the fungi. J. Biol. Chem., 259, 8033–8036. [PubMed] [Google Scholar]
  17. [18]Oakeley E.J. (1999) DNA methylation analysis: a review of current methodologies. Pharmacol. Ther., 84, 389–400. [DOI] [PubMed] [Google Scholar]
  18. [19]Belinsky S.A., Nikula,K.J., Baylin,S.B. and Issa,J.P. (1996) Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer. Proc. Natl Acad. Sci. USA, 93, 4045–4050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. [20]Hwu H.R., Roberts,J.W., Davidson,E.H. and Britten,R.J. (1986) Insertion and/or deletion of many repeated DNA sequences in human and higher ape evolution. Proc. Natl Acad. Sci. USA, 83, 3875–3879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. [21]Gu Z., Wang,H., Nekrutenko,A. and Li,W.H. (2000) Densities, length proportions and other distributional features of repetitive sequences in the human genome estimated from 430 megabases of genomic sequence. Gene, 259, 81–88. [DOI] [PubMed] [Google Scholar]
  21. [22]Kazazian H.H. Jr and Goodier,J.L. (2002) LINE drive. retrotransposition and genome instability. Cell, 110, 277–280. [DOI] [PubMed] [Google Scholar]
  22. [23]Kochanek S., Renz,D. and Doerfler,W. (1993) DNA methylation in the Alu sequences of diploid and haploid primary human cells. EMBO J., 12, 1141–1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. [24]Schmid C.W. (1998) Does SINE evolution preclude Alu function? Nucleic Acids Res., 26, 4541–4550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. [25]Bestor T.H. (1998) The host defence function of genomic methylation patterns. Novartis Found. Symp., 214, 187–195; discussion 195–199, 228–232. [DOI] [PubMed] [Google Scholar]
  25. [26]Rideout W.M. 3rd, Coetzee,G.A., Olumi,A.F. and Jones,P.A. (1990) 5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes. Science, 249, 1288–1290. [DOI] [PubMed] [Google Scholar]
  26. [27]Ronaghi M., Nygren,M., Lundeberg,J. and Nyren,P. (1999) Analyses of secondary structures in DNA by pyrosequencing. Anal. Biochem., 267, 65–71. [DOI] [PubMed] [Google Scholar]
  27. [28]Robertson K.D., Ait-Si-Ali,S., Yokochi,T., Wade,P.A., Jones,P.L. and Wolffe,A.P. (2000) DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nature Genet., 25, 338–342. [DOI] [PubMed] [Google Scholar]

Cited by

Sorted by citations

Authors
Yang, A. S.
Affiliations
The University of Texas MD Anderson Cancer Center (US)
Corresponding author
Yang, A. S.
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2004-02-13
Volume
32
Issue
3
Pages
38e-38
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gnh032
PMC
PMC373427
OpenAlex
W2106401558
Cited by
1,033
References
1
Citations by year
2026: 15 · 2025: 18 · 2024: 21 · 2023: 41 · 2022: 44 · 2021: 31 · 2020: 34 · 2019: 40 · 2018: 52 · 2017: 72 · 2016: 79 · 2015: 67 · 2014: 76 · 2013: 67 · 2012: 72
FWCI
9.49
Type
journal article
Language
en
Open access
Locations
  • Nucleic Acids Research (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Acervo Digital da Universidade Estadual Paulista (Universidade Estadual Paulista) (submittedVersion, other-oa)
  • Americanae (AECID Library) (submittedVersion, other-oa)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (submittedVersion, other-oa)
Funders
National Cancer Institute (P50CA100632, CA-16672, R33CA89837, R33CA089837, P30CA016672)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Syndromes and Imprinting
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
global DNA methylation, repetitive DNA, Alu elements, CpG methylation, decitabine, pyrosequencing, restriction enzyme digestion
MeSH terms
Decitabine, Azacitidine, DNA, Humans, Mutation, Repetitive Sequences, Nucleic Acid, Sensitivity and Specificity, Sulfites, Reproducibility of Results, Polymerase Chain Reaction, Sequence Analysis, DNA, CpG Islands, DNA Methylation, Long Interspersed Nucleotide Elements, Alu Elements, Cell Line, Tumor

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review