Detection Of DNA Modifications Research Articles

DNA Methylation and Susceptibility to Autism Spectrum Disorder.

The prevalence of autism spectrum disorder (ASD) has been increasing steadily over the last 20 years; however, the molecular basis for the majority of ASD cases remains unknown. Recent advances in next-generation sequencing and detection of DNA modifications have made methylation-dependent regulation of transcription an attractive hypothesis for being a causative factor in ASD etiology. Evidence for abnormal DNA methylation in ASD can be seen on multiple levels, from genetic mutations in epigenetic machinery to loci-specific and genome-wide changes in DNA methylation. Epimutations in DNA methylation can be acquired throughout life, as global DNA methylation reprogramming is dynamic during embryonic development and the early postnatal period that corresponds to the peak time of synaptogenesis. However, technical advances and causative evidence still need to be established before abnormal DNA methylation and ASD can be confidently associated.

Vertical Ultrafiltration-Facilitated DNA Digestion for Rapid and Sensitive UHPLC-MS/MS Detection of DNA Modifications.

LC-MS/MS technologies provide important and powerful analytical tools for chemical structure-dependent identification and quantification of epigenetically crucial DNA modifications. To perform LC-MS/MS analysis, it is better to convert DNA to 2'-deoxynucleosides through enzymatic digestion. Here, we observed that inorganic cations Na+ and K+ and phosphate buffers, which were often found in various DNA solutions, significantly inhibited DNA digestion as catalyzed by typical set of DNase I, snake venom phosphodiesterase, and calf intestine alkaline phosphatase, leading to poor or varying performance on UHPLC-MS/MS analysis. We then developed an efficient and unique vertical-ultrafiltration approach, enabling usto remove these inorganic salts without DNA loss. Consequently, the removal of inorganic salts by ultrafiltration facilitated the followed DNA digestion and thus enhanced the final UHPLC-MS/MS detection. Benefiting from the developed vertical-ultrafiltration approach, it is also feasible to integrate the desalting step with the other two steps of DNA digestion and protein removal. By investigating the time course of DNA digestion, we observed a differential release rate of 2'-deoxycytidine and 5-methyl-2'-deoxycytidine causing a measurement bias on the methylation frequency. We further exploited Mg2+ to eliminate this bias by stimulating DNase set-based DNA digestion. These innovative approaches enable us to perform rapid, sensitive, and robust UHPLC-MS/MS analysis of methylated DNA 2'-deoxycytidine, demethylation intermediates, and probably other DNA modifications.

Early detection of staurosporine-induced apoptosis by comet and annexin V assays.

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction.

Detection of DNA modifications by the 32P-postlabelling assay

The 32P-postlabelling assay is widely used for detection of carcinogen–DNA adducts and other types of modified nucleotides in DNA. The principle of the method is the enzymatic digestion of DNA to nucleotides, 5′-labelling of these nucleotides with an isotopically labelled phosphate group, and the resolution and detection of the labelled products. Since the development of the original procedure in the early 1980s, many methods have been developed to increase the sensitivity of the method by selection of the modified nucleotides prior to labelling. In favourable circumstances, the method can achieve a level of detection as low as 1 modification in 10 10 nucleotides and requires relatively small quantities of DNA (less than 10 μg). It has been used to detect and characterise DNA adducts formed by numerous genotoxic carcinogens in bacterial and mammalian cells, in animals and, in some cases, in human tissues. Most classes of carcinogen have been subjected to 32P-postlabelling analysis, ranging from bulky and/or aromatic compounds to small and/or aliphatic compounds; it has also been used, with modifications, to detect apurinic sites in DNA, oxidative damage to DNA, UV-induced photodimers and, to a lesser extent, DNA damage caused by cytotoxic drugs. It has provided the first clear evidence for the DNA-damaging properties of several synthetic carcinogenic hormones. It has revealed the DNA-damaging potential of complex mixtures such as coal-tar and tobacco smoke. It has been used in human biomonitoring studies to detect DNA damage from occupational exposure to carcinogens, and also from environmental (i.e. non-occupational) exposures. It has also led to the discovery of the presence of numerous modifications in DNA arising from endogenous processes. The rapid expansion in the use of the assay has resulted in some divergence of procedures and there is a case to be made for the use of more standardised protocols, particularly where human exposure to carcinogens is being measured and where such results may be required for risk assessment. While the procedure is quantifiable, the efficiency of adduct labelling is, in many cases, not quantitative, and the lack of adduct standards has, in many cases, limited the interpretation of data to a demonstration of higher adduct levels in exposed groups compared with unexposed groups. Future developments are expected in automation, standardisation and, in combination with other analytical methods, elucidation of the structures of the many DNA lesions whose existence has been revealed by the 32P-postlabelling technique.

Mass-Spectrometric Approaches for DNA-Based Genetic Screening

In the current issue of Clinical Chemistry , Braun et al. (1) describe a mutational analysis method that combines pr imer o ligo b ase e xtension (PROBE) and m atrix- a ssisted l aser d esorption i onization t ime- o f- f light m ass s pectrometry (MALDI-TOF-MS). This application further extends the use of MS for genetic analysis and suggests a possible role in the clinic. However, what is the long-term potential for MALDI, based on the relative strengths and weaknesses of MS analysis of DNA (2)? The development of soft-ionization techniques for MS has, without question, revolutionized protein chemistry. MS is now one of the definitive methods for characterization of synthetic and isolated peptides and proteins. In addition to accurately determining an intact mass, one can obtain primary structure information by several different MS sequencing strategies, and secondary and tertiary structure can be evaluated by hydrogen-exchange experiments. The use of MS for DNA analysis, although first demonstrated for nucleosides in 1962 (3), is far less advanced. Potential applications of MS are (a) detection of DNA modifications, (b) DNA fragment mass determination, and (c) DNA sequencing. Both fast atom bombardment (FAB) and electrospray ionization (ESI) collision-induced dissociation/tandem MS have been applied for identification of DNA modification sites. For example, the …