Methylation-dependent fragment separation: Direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA

Boyd, Victoria L.; Moody, Kristina I.; Karger, Achim E.; Livak, Kenneth J.; Zon, Gerald; Burns, John W.

doi:10.1016/j.ab.2006.04.009

Cited by 33 publications

(24 citation statements)

References 28 publications

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“…A procedure for quantification of methylation of cytosine in genomic DNA was reported (106). First, a treatment with bisulfite selectively deaminated cytosine (to yield thymine) but not 5-methylcytosine; second, the forward strands are amplified by PCR using a fluorescently labeled primer.…”

Section: Applications Nucleic Acid Analysismentioning

confidence: 99%

Capillary Electrophoresis in Bioanalysis

2008

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Science reports ∼4800 hits on capillary electrophoresis (CE) including 410 reviews. Because of the prominence of CE techniques in bioanalysis, we decided make them the focus of this review and selected 200 hundred papers representing advances in this field. The selected papers cover advances in CE theory, instrumentation, and methodologies that are specific to various analytes of biological origin or relevance. The group of analytes includes nucleic acids, proteins and peptides, carbohydrates, lipids, single cells, and bioparticles. In addition, we have included advances in the use of CE to define functional assays or to investigate biomolecular interactions. The use of microfabricated devices for CE analysis was not included because this is already covered in other review. Technique Developments Separation SchemesDetermining the velocity of the electroosmotic flow (EOF) and how it changes during an electrophoretic separation is still an important research topic. A simple method for EOF measurements using so-called thermal marks was reported (1). Here, a tungsten filament caused punctual heating at the capillary wall and caused a perturbation in the electrolyte concentration. A sequence of these "thermal marks" then migrated with the EOF until each mark reached and was detected by a conductivity detector. The feasibility of using thermal marks as internal EOF standards in different separation systems was thereby demonstrated.Isoelectric focusing separates amphoteric analytes such as proteins or peptides by the differences in their isoelectric points. Most of the reports on capillary isoelectric focusing (cIEF) describe an initial focusing phase after which the focused zones are mobilized and detected. A dynamic cIEF method for protein analysis was reported (2). This technique made it possible to control each protein's position and focused width by moving the pH gradient within the capillary through manipulation of the electric fields. An important advantage of this approach is the capability of collecting focused analytes from the central section, suggesting that there may be great potential for introducing selectively focused proteins to a second separation dimension such as LC or CE.Micellar electrokinetic capillary chromatography (MEKC) is typically incompatible with electrospray mass spectrometry (ESI-MS) because the nonvolatile surfactants in the micellar phase result in complicated adduct formation and loss of sensitivity during the electrospray process and because the presence of the organic solvent needed for electrospray may cause instability in the micellar phase. These drawbacks were overcome by using synthetic polymeric surfactants that can work as a pseudostationary phase and provide stable electrospray (3). The polymeric surfactant was made by polymerizing three amino acidderived (L-leucinol, L-isoleucinol, L-valinol) sulfated chiral surfactants. These polymeric

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Section: Applications Nucleic Acid Analysismentioning

confidence: 99%

Capillary Electrophoresis in Bioanalysis

2008

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“…The heterogeneous methylation of the hMLH1 promoter was identified in samples from many colorectal cancer patients, in which hMLH1 promoter methylation was found to have a significant relationship to the loss of protein expression and to increase with the age of the patient [39]. Several DNA methylation studies of this kind have also been reported by others researchers [40,41].…”

Section: Ce In Genomicsmentioning

confidence: 79%

CE at the omics level: Towards systems biology – An update

Song

Babar

et al. 2007

Electrophoresis

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This review provides an updated overview of recent developments and applications of CE based on previously published reports in the field of omic research. The increased number of published articles on omics shows that the field is growing and attracting the attention of many life science researchers. Due to developments in the omics sciences, many researchers have been studying systems biology, in which biological events in organisms are systematically interpreted through the combination of complex measurements from various methods resulting in high-throughput data. Given the challenges of such complex forms of analysis, CE is a strong candidate for generating omics data useful for acquiring the qualitative and quantitative knowledge necessary for systems-level investigation. By emphasizing CE for systems biology, this review will discuss and focus on the applicability of CE to systems-based analytical data at the genomic, transcriptomic, proteomic, and metabolomic levels from 2005 to the present.

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“…This difference in migration speed could be due to the variation in the base composition of Met and nonMet alleles or to the degradative effect of sodium bisulfite on DNA. Reports on differential migration of Met and nonMet DNA on sodium bisulfite treatment are available in this context (Zhou et al, 2004;Boyd et al, 2006). (b) In the current study, 28 (39.29%) was the most common CGG repeat observed followed by 27 (24.04%).…”

Section: Validationmentioning

confidence: 81%

“…(d) There appears to be a considerable level of allele dropout using the rPCR method, that is, the preferential amplification of only one allele (normal), dropping out the larger/expanded allele from amplification. (e) As Boyd et al (2006) pointed; it would not be easy to apply a correction factor, because larger amplicons deviate from the correction factor predictions for smaller fragments, possibly due to secondary structure formation. However, if necessary, a correction factor of + 2 for alleles with Ms-PCR nonMet sizing of up to 35 repeats, and + 3 for Ms-PCR nonMet sizes of 36-54 repeats could be considered.…”

Section: Validation Result-2mentioning

confidence: 99%

Fragile X CGG Repeat Variation in Tamil Nadu, South India: A Comparison of Radioactive and Methylation-Specific Polymerase Chain Reaction in CGG Repeat Sizing

Nagarathinam

Singh²,

Chong³

et al. 2012

Genetic Testing and Molecular Biomarkers

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Fragile X syndrome is the most frequent hereditary cause of mental retardation after Down syndrome. Expansion of CGG repeats in the 5¢ UTR of the fragile X mental retardation gene 1 (FMR1) causes gene inactivation in most of the cases. The FMR1 gene is classified into normal 5-44; gray zone 45-54; premutation 55 to < 200; and full mutation ‡ 200 repeats. Precise sizing of FMR1 alleles is important to understand their variation, predisposition, and for genetic counseling. Meta-analysis reveals prevalence of premutation carriers as 1 in 259. No such reports are available in India. About 705 women from Tamil Nadu, South India, were screened for the FMR1 allelic variation by using radioactive polymerase chain reaction-polyacrylamide gel electrophoresis (PAGE) analysis. The women who were homozygous by radioactive polymerase chain reaction (rPCR) were reanalyzed by methylation-specific polymerase chain reaction (Ms-PCR) and GeneScan analysis. The techniques were validated and compared to arrive at a correction factor. Among 122 genotypes, 35 repeat variants ranging in size from 16 to 57 were observed. The most common repeat is 30 followed by 29. One in 353 women carried the premutation. No full mutations were observed. Screening populations with low frequency of premutations may not be applicable. Ms-PCR is more suitable for routine screening and clinical testing compared with rPCR-PAGE analysis.

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Methylation-dependent fragment separation: Direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA

Cited by 33 publications

References 28 publications

Capillary Electrophoresis in Bioanalysis

Capillary Electrophoresis in Bioanalysis

CE at the omics level: Towards systems biology – An update

Fragile X CGG Repeat Variation in Tamil Nadu, South India: A Comparison of Radioactive and Methylation-Specific Polymerase Chain Reaction in CGG Repeat Sizing

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