Detection Of Cytosine Research Articles

A Capillary Electrophoresis UV Detection-Based Method for Global Genomic DNA Methylation Assessment in Human Whole Blood.

Quantitative analysis of DNA methylation patterns is of special importance in several developmental and pathological situations. The development of simple and robust methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly reproducible CE-UV method for the separation and detection of cytosine and methylcytosine, after formic acid hydrolysis of DNA extracted from human whole blood. Hydrolyzed samples were dried and successively dissolved with water and then injected into the capillary without sample derivatization procedures. The use of a run buffer containing 50mmol/L BIS-TRIS propane (BTP) phosphate buffer at pH3.25 and 60mmol/L sodium acetate buffer at pH3.60 (4:1, v/v) allowed a baseline analytes separation within 12min. Precision tests indicated an elevated reproducibility with an inter-assay CV of 1.98%.

Detection of cytosine and CpG density in proto-oncogenes and tumor suppressor genes in promoter sequences of acute myeloid leukemia

ABSTRACTAberrant methylation is one of the driving forces of cancer genome development. Although the rate of methylation appears massively variable across the genome, it is mainly observed in histone modification, chromatin organization, DNA accessibility, or promoter sequence. Methylation of promoter sequence occurs mostly to cytosine nucleotides, which can affect transcription factors' binding affinities. In this study, we demonstrated that cytosine repeats (C types density), consisting of CC, CCC, CCCC, CCCCC, CCCCCC, CCCCCCC motifs and CpG islands density in 25 proto-oncogenes, tumor suppressor genes and control genes may play a role in the pathogenesis of acute myeloid leukemia. The promoter sequences were divided into a 100 nucleotide window from −500 to +100 nucleotides and 20 nucleotide window from −100 to +100. Each window is analyzed to find the higher C type and CpG islands density, which may cause the increased methylation in the promoter sequence. Our novel findings show that promoter sequence cytosine repeats and CpG density increase closer to transcription sites, especially just before and after the transcription start site (TSS). The results demonstrate that cytosine density increases while proto-oncogenes and TSG promoter sequences are closer to TSS 50.8% and 41.0% respectively, if (−500 to −200) and (−100 to +100) windows of the nucleotide sequences are compared. This proves that around TSS location has special nucleotide motifs and could be an important implication for our understanding of potential methylating locations in promoters.

Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection.

Alterations in global DNA methylation are implicated in various pathophysiological processes. The development of simple and quick, yet robust, methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly sensitive and reproducible capillary electrophoresis method with UV detection for the separation and detection of cytosine and methylcytosine, after formic acid hydrolysis of DNA extracted from human whole blood. Hydrolysed samples were dried and resuspended with water and directly injected into the capillary without sample derivatization procedures. The use of a run buffer containing 50 mmol/L BIS-TRIS propane (BTP) phosphate buffer at pH 3.25 and 60 mmol/L sodium acetate buffer at pH 3.60 (4 : 1, v/v) allowed full analyte identification within 11 min. Precision tests indicated an elevated reproducibility with an interassay CV of 1.98% when starting from 2 μg of the extracted DNA. The method was successfully tested by measuring the DNA methylation degree both in healthy volunteers and in reference calf thymus DNA.

Paramagnetic Particles Isolation of Influenza Oligonucleotide Labelled with CdS QDs

In this study, we describe hybridization design probes consisting of paramagnetic particles and quantum dots (QDs) with targeted DNA, and their application for detection of avian influenza virus (H5N1). Optical properties of QDs were beneficial, but the main attention was paid to the electroactivity of metal part of QDs and ODNs themselves. Differential pulse voltammetry was used for detection of cadmium(II) ions and square wave voltammetry for detection of cytosine–adenine peak in ODN-SH-Cd complex. It clearly follows from the obtained results that the optimized conditions were temperature of hybridization 25 °C, time of hybridization 35 min, and concentration of ODN-SH-Cd complex 20 μg mL−1. The detection limit (3 signal/noise) was estimated as 15 ng mL−1 of ODN-SH-Cd.

The use of cytosine as a marker to estimate microbial protein synthesis in the rumen

Recently methods based upon the HPLC separation and detection of cytosine (Koenig 1980) and the spectrophotometric analysis of the total purine content of microbes (Zinn and Owens 1986) have been proposed to estimate microbial nitrogen flow at the duodenum. Little work has been undertaken in-vivo to evaluate cytosine as a marker and to compare it with current techniques. The present trial was designed to evaluate this marker and to compare it with diaminopimelic acid (DAPA), [3H]leucine and the total purine technique. In addition, recent reports (Hvelpund and Madsen 1985) have indicated that expressing miaobial efficiency in terms of carbohdrate, as opposed to organic matter degraded in the rumen, reduced the variation in microbial yield. This was also investigated.

Detection of Drug-Induced DNA Hypermethylation in Human Tumor Cells Exposed to Cancer Chemotherapy Agents

Abstract A method is described for the quantitative analysis of drug-induced DNA hypermethylation in human tumor cells exposed in vivo or in vitro to DNA synthesis-inhibiting levels of cancer chemotherapy agents. The method relies upon isocratic separation of formic acid hydrolysates of DNA using an Aminex A-9 column converted to the cation form. When combined with pre-incorporation of uridine or deoxycytidine labeled specifically in the 6-position, the method is sensitive in the picomolar range for 5-methyl-cytosine, and is specific for the detection of cytosines methylated during the period of drug-induced DNA synthesis inhibition. The method can be used to quantitate DNA hypermethylation occurring in patient bone marrow or peripheral blood specimens in vivo during chemotherapy.