Bisulfite Sequencing of DNA

Darst, Russell P.; Pardo, Carolina E.; Ai, Lingbao; Brown, Kevin D.; Kladde, Michael P.

doi:10.1002/0471142727.mb0709s91

Cited by 84 publications

(58 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Genomic DNA harvested from cultured cell lines was bisulfite converted using an in-house protocol as described previously (33). Polymerase chain reaction (PCR) amplification of the lower DNA strand was conducted in a 20 ll reaction volume containing 1Â Coral buffer (Qiagen), 250 nM deoxynucleotides, 250 nM each of forward (5#-GtAGGtTttTTGGtAtttAGGt-3#; lower case indicating C to T transitions) and reverse (5#-CATaCTaCTCAaaACCTCCT-3#; lower case indicating G to A transitions) primer, 1 U of HotStarTaq Plus DNA polymerase (Qiagen) and 2 ll (50-100 ng) of bisulfite-treated DNA.…”

Section: Pyrosequencing Analysismentioning

confidence: 99%

“…no enzyme control) were done as described previously (36). Purified genomic DNA was subjected to bisulfite genomic sequencing (37) as described in (33). A 712 bp region of the WIF1 promoter was PCR amplified by forward (5#-TATATACTCGAGATtAttATtAttATtATtAGYAtTtAGTt-3#; XhoI site underlined; where Y is a pYrimidine and lower case indicates C to T transitions) and reverse (5#-ATATATAAGCTTCAaRCACAaaAaaATRCTCCAaA-3#; HindIII site underlined; where R is a puRine and lower case indicates G to A transitions) primers.…”

Section: Methyltransferase Accessibility Protocol For Individual Tempmentioning

confidence: 99%

See 1 more Smart Citation

WIF1 is a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix

et al. 2011

Self Cite

View full text Add to dashboard Cite

Aberrant activation of the Wnt/b-catenin signaling axis is a prominent oncogenic mechanism in numerous cancers including cervical cancer. Wnt inhibitory factor-1 (WIF1) is a secreted protein that binds Wnt and antagonizes Wnt activity. While the WIF1 gene is characterized as a target for epigenetic silencing in some tumor types, WIF1 expression has not been examined in human cervical tissue and cervical cancer. Here, we show that WIF1 is unmethylated and its gene product is expressed in normal cervical epithelium and some cultured cervical tumor lines. In contrast, several cervical cancer lines contained dense CpG methylation within the WIF1 gene, and expression of both WIF1 transcript and protein was restored by culturing cells in the presence of the global DNA demethylating agent 5-aza-2#-deoxycytidine. Using single-molecule MAPit methylation footprinting, we observed differences in chromatin structure within the WIF1 promoter region between cell lines that express and those that do not express WIF1, consistent with transcriptional activity and repression, respectively. The WIF1 promoter was aberrantly methylated in $60% (10 of 17) high-grade highly undifferentiated squamous cell cervical tumors examined, whereas paired normal tissue showed significantly lower levels of CpG methylation. WIF1 protein was not detectable by immunohistochemistry in tumors with quantitatively high levels of WIF1 methylation. Of note, WIF1 protein was not detectable in two of the seven unmethylated cervical tumors examined, suggesting other mechanisms may contribute WIF1 repression. Our findings establish the WIF1 gene as a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix. Background

show abstract

Section: Pyrosequencing Analysismentioning

confidence: 99%

Section: Methyltransferase Accessibility Protocol For Individual Tempmentioning

confidence: 99%

WIF1 is a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix

et al. 2011

Self Cite

View full text Add to dashboard Cite

show abstract

“…The treatment of DNA with sodium bisulfite (Na + HSO 3 -) converts unmethylated cytosines to uracils, while methylated cytosines are not affected (Darst et al, 2010). DNA is fragmented (~200-800 bp) by sonication and is treated with bisulfite at lower pH (pH 5) which adds sulfite group to the C6 of cytosine, which is then followed by incubating the samples at higher pH, which removes sulfite group generating uracil.…”

Section: Bisulfite Sequencing and Methylation-specific Pcr (Msp)mentioning

confidence: 99%

Crime investigation through DNA methylation analysis: methods and applications in forensics

Rana¹

2018

Egypt J Forensic Sci

View full text Add to dashboard Cite

Discerning monozygotic twins, precisely predicting the age of human beings and most importantly interpreting human behavior remains a big challenge in forensic science. Modern advances in the field of epigenetics especially analysis of DNA methylation pattern can certainly help to identify and discriminate various single-source DNA samples such as bodily fluids collected from the crime scene. In this review, cytosine methylation analysis of DNA has been methodologically discussed with a broad range of applications in forensic science such as identifying single or mixed body fluid, discriminating monozygotic twins, age prediction, addiction/behavioral prediction as well as race/ethnicity identification. With the advances of next-generation sequencing techniques, blooming of DNA methylation datasets and together with standard epigenetic protocols, the prospect of investigating and solving these critical issues in forensic science is highly promising in the court of law.

show abstract

“…In the BS method, bisulfite-treated DNA is PCR-amplified with methylation-independent primers (methylation-specific PCR or MSP) and size-fractionated via gel electrophoresis (Darst et al, 2010;Herman et al, 1996). The products purified by PCR were cloned into Escherichia coli, and five to ten individual clones were sequenced.…”

Section: Analysis Of Epigenetic Mechanisms Through Dna Methylationmentioning

confidence: 99%

Environmental epigenomics: Current approaches to assess epigenetic effects of endocrine disrupting compounds (EDC’s) on human health

Tapia-Orozco

Santiago-Toledo

Barrón

et al. 2017

Environmental Toxicology and Pharmacology

View full text Add to dashboard Cite

Roeb, Environmental epigenomics: Current approaches to assess epigenetic effects of endocrine disrupting compounds (EDC's) on human health.Environmental Toxicology and Pharmacology http://dx.doi.org/10. 1016/j.etap.2017.02.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. AbstractEnvironmental Epigenomics is a developing field to study the epigenetic effect on human health from exposure to environmental factors. Endocrine disrupting chemicals have been detected primarily in pharmaceutical drugs, personal care products, food additives, and food containers. Exposure to endocrine-disrupting chemicals (EDCs) has been associated with a high incidence and prevalence of many endocrine-related disorders in humans.Nevertheless, further evidence is needed to establish a correlation between exposure to EDC and human disorders.Conventional detection of EDCs is based on chemical structure and concentration sample analysis. However, substantial evidence has emerged, suggesting that cell exposure to EDCs leads to epigenetic changes, independently of its chemical structure with nonmonotonic low-dose responses.Consequently, a paradigm shift in toxicology assessment of EDCs is proposed based on a comprehensive review of analytical techniques used to evaluate the epigenetic effects.Fundamental insights reported elsewhere are compared in order to establish DNA methylation analysis as a viable method for assessing endocrine disruptors beyond the conventional study approach of chemical structure and concentration analysis.

show abstract

Bisulfite Sequencing of DNA

Cited by 84 publications

References 41 publications

WIF1 is a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix

WIF1 is a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix

Crime investigation through DNA methylation analysis: methods and applications in forensics

Environmental epigenomics: Current approaches to assess epigenetic effects of endocrine disrupting compounds (EDC’s) on human health

Contact Info

Product

Resources

About