Direct potential resolution and simultaneous detection of cytosine and 5-methylcytosine based on the construction of polypyrrole functionalized graphene nanowall interface

Wang, Po; Han, Peng; Dong, Liang; Miao, Xiangshui

doi:10.1016/j.elecom.2015.09.025

Cited by 33 publications

(9 citation statements)

References 55 publications

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“…Interestingly, a comparison between the electrochemical methods reported so far for the determination of different types of targets (synthetic oligonucleotides, free DNA bases, PCR products and genomic DNA), demonstrates that the LODs achieved with the DNA sensor for the synthetic short target methylated DNAs are much lower than those reported with PCR amplification (25 pg) 11 , as well as for PCR-free methods using direct oxidation of DNA bases of short methylated oligonucleotides (0.11 μM) 35 and free un-methylated C (0.6 μM) 38 , digestion by a restriction enzyme of a synthetic target DNA (10 nM) 27 , bisulfite conversion of synthetic target DNAs (18 pM) 34 and PCR products (0.5 nM) 37 , and a paired-end tagging and amplification electrochemical strategy for methylated genomic DNA (40 pg) 23 . It is important to note also that the most sensitive strategies among these required assay times between 1.5 and 24.5 h 11 , 23 , 34 compared to the 1 h of the DNA sensors developed in this work.…”

Section: Resultsmentioning

confidence: 95%

Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments

Povedano

Vargas

Montiel

et al. 2018

Sci Rep

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This paper describes two different electrochemical affinity biosensing approaches for the simple, fast and bisulfite and PCR-free quantification of 5-methylated cytosines (5-mC) in DNA using the anti-5-mC antibody as biorecognition element. One of the biosensing approaches used the anti-5-mC as capture bioreceptor and a sandwich type immunoassay, while the other one involved the use of a specific DNA probe and the anti-5-mC as a detector bioreceptor of the captured methylated DNA. Both strategies, named for simplicity in the text as immunosensor and DNA sensor, respectively, were implemented on the surface of magnetic microparticles and the transduction was accomplished by amperometry at screen-printed carbon electrodes by means of the hydrogen peroxide/hydroquinone system. The resulting amperometric biosensors demonstrated reproducibility throughout the entire protocol, sensitive determination with no need for using amplification strategies, and competitiveness with the conventional enzyme-linked immunosorbent assay methodology and the few electrochemical biosensors reported so far in terms of simplicity, sensitivity and assay time. The DNA sensor exhibited higher sensitivity and allowed the detection of the gene-specific methylations conversely to the immunosensor, which detected global DNA methylation. In addition, the DNA sensor demonstrated successful applicability for 1 h-analysis of specific methylation in two relevant tumor suppressor genes in spiked biological fluids and in genomic DNA extracted from human glioblastoma cells.

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Section: Resultsmentioning

confidence: 95%

Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments

Povedano

Vargas

Montiel

et al. 2018

Sci Rep

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“…90 Direct electrochemical oxidation of mC is also a useful detection method. 91,92 A number of other methods based on electrochemical detection, [93][94][95][96] and FRET based methods for the detection of mC have also been reported. [97][98][99] Derivatisation of DNA with O-allylhydroxylamine has been used by Carell and co-workers to detect mC.…”

Section: Chemical Methods Beyond Bisulfite Sequencingmentioning

confidence: 99%

Methods for detection of cytosine and thymine modifications in DNA

Berney

McGouran

2018

Nat Rev Chem

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Methylation of cytosine at the 5-position is a common epigenetic marker in mammalian DNA, and plays an important role in regulating gene expression. Oxidised derivatives of 5-methylcytosine have recently been discovered. As well as being intermediates in an active demethylation pathway, some of these oxidised derivatives may function as epigenetic markers in their own right. Oxidised derivatives of thymine are also known as products of DNA damage. There is evidence however that one such derivative, 5-hydroxymethyluracil, may play an epigenetic role. There is a pressing need to learn more about these modifications, due to the role epigenetic markers play in development, and diseases such as cancer. This emerging area of research requires methods for detecting cytosine and thymine modifications in DNA with a high degree of accuracy and sequence specificity. This review will introduce the biochemistry of cytosine and thymine modifications, and discuss new and established detection methods which have been developed to overcome the high degree of difficulty associated with studying these modifications in DNA. Introduction: Cytosine and Thymine Modifications DNA codes for all the proteins necessary for life, but the DNA sequence is not the sole determinant of all the phenotypic traits of cells and organisms. Transcription of DNA is tightly regulated by epigenetic modifications, which play an important role in controlling when and where specific genes are expressed. 1,2 Epigenetic modifications regulate important processes such as cellular differentiation, and are also implicated in various diseases including cancer, autism spectrum disorder, and other developmental diseases such as Rett syndrome. 3-5 Epigenetic control of gene expression is often mediated through covalent modification of DNA itself, or of the histone proteins which pack DNA in the nucleus. These modifications do not result in changes to the DNA sequence, but can affect the binding of certain proteins to the DNA, including transcription factors and proteins which regulate chromatin structure. 6-8 In mammals and many other eukaryotes, the most common epigenetic covalent modification of DNA is methylation of cytosine (C) at the 5-position. Such modifications can be inherited, but can also be enzymatically introduced and removed in response to stimuli. 9 5-Methylcytosine (mC) is introduced by the methylation of cytosine residues by DNA-methyltransferases (DNMTs) with an S-adenosylmethionine cofactor. 5-Methylcytosine is most often found in the context of 5'-cytosinephosphate-guanine-3' (CpG) dinucleotides. Regions containing a high density of CpG sequences are found in about 72% of promoters in the human genome. 10 Methylation of these regions causes

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“…PNA (peptide Bylo rovněž publikováno několik strategií využívajících elektrochemickou analýzu metylace DNA. Nejjednodušší je přímé rozlišení cytozinu a metylcytozinu na povrchu určitých elektrod na bázi uhlíku, a to díky rozdílnému potenciálu jejich oxidace [43]. Výsledná voltametrická křivka (křivka závislosti proudu na potenciálu) pak obsahuje dva oddělené píky, jeden od cytozinu a druhý od metylcytozinu.…”

Section: Závěr a Výhled Do Budoucnostiunclassified

Novel Approaches in DNA Methylation Studies – MS-HRM Analysis and Electrochemistry

Bartošík¹,

Ondroušková²

2016

Klin Onkol

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Regionální centrum aplikované molekulární onkologie, Masarykův onkologický ústav, Brno Souhrn Metylace cytozinů v DNA je jedním z epigenetických mechanizmů regulujících expresi genů, a hraje tak důležitou roli v diferenciaci nebo proliferaci buněk. V nádorových buňkách často dochází ke změnám v metylaci DNA, kupříkladu nadměrnou metylací (hypermetylací) promotorů tumor supresorových genů. Proto jsou vyvíjeny nové metody analýzy, které by byly schopny určit rozsah metylace konkrétní sekvence DNA. K těmto metodám se řadí i relativně levné a rychlé techniky MS-HRM (methylation-specific high resolution melting) a elektrochemie. Ve srovnání s jinými používanými metodami jsou vhodné pro screening většího počtu vzorků či více cílových oblastí DNA. MS-HRM svou citlivostí a relativní nenáročností konkuruje ostatním zavedeným metodám, jako jsou metylačně-specifická PCR nebo přímá bisulfitová sekvenace. Elektrochemie nabízí nenáročné přístrojové vybavení a při použití vhodných elektroaktivních molekul a elektrodových povrchů i několik zajímavých strategií rozlišení cytozinů a metylcytozinů. Obě techniky byly již úspěšně použity při stanovení metylace DNA promotorů důležitých tumor supresorových genů a mohly by tak v budoucnu přispět ke zpřesnění dia gnostiky a prognostiky onkologických onemocnění. Aberantní metylace promotorů byla popsána již u stovek genů se vztahem k nádorovým onemocněním a s rozvojem metod, jež by umožňovaly jejich detekci i v klinické praxi, by se řada z nich mohla stát novými důležitými bio markery. Klíčová slova DNA metylace-5-metylcytozin-HRM analýza-teplota tání-DNA duplex-elektrochemiehybridizace nukleových kyselin Summary Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and meth ylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.

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Direct potential resolution and simultaneous detection of cytosine and 5-methylcytosine based on the construction of polypyrrole functionalized graphene nanowall interface

Cited by 33 publications

References 55 publications

Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments

Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments

Methods for detection of cytosine and thymine modifications in DNA

Novel Approaches in DNA Methylation Studies – MS-HRM Analysis and Electrochemistry

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