An overview of the analysis of DNA methylation in mammalian genomes

DNA methylation at position C5 of the pyrimidine ring of cytosine in mammalian genomes has received a great deal of research interest due to its importance in many biological phenomena. It is associated with events such as epigenetic gene silencing and the maintenance of genome integrity. Aberrant DNA methylation, particularly that of chromosomal regions called CpG islands, is an important step in carcinogenesis. In order to elucidate methylation profiling of complex genomes, various methods have been developed. Many of these methods are based on the differential reactivity of cytosine and 5-methylcytosine to various chemicals. The combined use of these chemical reactions and other preexisting methods has enabled the discrimination of cytosine and 5-methylcytosine in complex genomes. The use of proteins that preferentially bind to methylated DNA has also successfully been used to discriminate between methylated and unmethylated sites. The chemical and structural dissection of the in vivo processes of enzymatic methylation and the binding of methyl-CpG binding proteins provides evidence for the complex mechanisms that nature has acquired. In this review we summarize the methods available for the discrimination between cytosine and 5-methylcytosine in complex genomes.     

表观遗传学新标记——5-羟甲基胞嘧啶检测方法的研究进展

被称为"第六种碱基"的5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC),广泛分布于多种哺乳动物的组织和细胞中,与胚胎发育,神经系统功能以及肿瘤研究高度相关.与5-甲基胞嘧啶(5-methylcytosine, 5mC)相比,5hmC在组织中含量更低,难以精确的检测.随着研究的深入,5hmC参与的重要生物学作用逐渐被人们发现,同时也促使着5hmC的检测和定量方法不断发展.为了区分5hmC与其他胞嘧啶衍生物,很多利用化学或者酶学修饰实现靶向检测或非靶向富集5hmC的方法应运而生.因此,选择并发展灵敏,准确,可靠的5hmC检测技术对于表观遗传研究至关重要.本文重点综述了近年来发展起来的5hmC检测和测序技术,通过比较分析各种方法的优缺点,为研究人员选择特定合适的方法开展相关研究提供重要的参考.     

Rapid DNA chemical ligation for amplification of RNA and DNA signal

Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.     

Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques

The epigenetic modification of 5-hydroxymethylcytosine (5hmC) is receiving great attention due to its potential role in DNA methylation reprogramming and as a cell state identifier. Given this interest, it is important to identify reliable and cost-effective methods for the enrichment of 5hmC marked DNA for downstream analysis. We tested three commonly used affinity-based enrichment techniques; (i) antibody, (ii) chemical capture and (iii) protein affinity enrichment and assessed their ability to accurately and reproducibly report 5hmC profiles in mouse tissues containing high (brain) and lower (liver) levels of 5hmC. The protein-affinity technique is a poor reporter of 5hmC profiles, delivering 5hmC patterns that are incompatible with other methods. Both antibody and chemical capture-based techniques generate highly similar genome-wide patterns for 5hmC, which are independently validated by standard quantitative PCR (qPCR) and glucosyl-sensitive restriction enzyme digestion (gRES-qPCR). Both antibody and chemical capture generated profiles reproducibly link to unique chromatin modification profiles associated with 5hmC. However, there appears to be a slight bias of the antibody to bind to regions of DNA rich in simple repeats. Ultimately, the increased specificity observed with chemical capture-based approaches makes this an attractive method for the analysis of locus-specific or genome-wide patterns of 5hmC.     

Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

Several approaches are used to survey genomic DNA methylation patterns, including Southern blot, PCR, and microarray strategies. All of these methods are based on the use of methylation-sensitive isoschizomer restriction enzyme pairs and/or sodium bisulfite treatment of genomic DNA. They have many limitations, including PCR bias, lack of comprehensive assessment of methylated sites, labor-intensive protocols, and/or the need for expensive equipment. Since the presence of 5-methylcytosine alters the melting properties of DNA molecules, denaturing gradient gel blots (DGG blots), a gene scanning technique which detects differences in DNA fragments based on differential melting behavior, were used to examine genomic modification patterns in normal tissues. Variations in melting behavior, observed as restriction fragment melting polymorphisms (RFMPs), were detected in various tissues from single individuals in all human and mouse genes tested, suggesting the presence of widespread differential cell type-specific DNA modification. Additional DGG blot experiments comparing genomic DNA to unmethylated cloned DNA suggested that the melting variants were most likely caused by DNA methylation differences. The results suggest that the use of DGG blots can provide a comprehensive and rapid method for comparing complex in vivo DNA modification patterns in normal adult somatic cells.     

On-line cell lysis of bacteria and its spores using a microfluidic biochip

Optimal detection of pathogens by molecular methods in water samples depends on the ability to extract DNA rapidly and efficiently. In this study, an innovative method was developed using a microfluidic biochip, produced by microelectrochemical system technology, and capable of performing online cell lysis and DNA extraction during a continuous flow process. On-chip cell lysis based on chemical/physical methods was performed by employing a sufficient blend of water with the lysing buffer. The efficiency of lysis with microfluidic biochip was compared with thermal lysis in Eppendorf tubes and with two commercial DNA extraction kits: Power Water DNA isolation kit and ForensicGEM Saliva isolation kit in parallel tests. Two lysing buffers containing 1% Triton X-100 or 5% Chelex were assessed for their lysis effectiveness on a microfluidic biochip. SYBR Green real-time PCR analysis revealed that cell lysis on a microfluidic biochip using 5% Chelex buffer provided better or comparable recovery of DNA than commercial isolation kits. The system yielded better results for Gram-positive bacteria than for Gram-negative bacteria and spores of Gram-positive bacteria, within the limits of detection at 10³ CFU/ml. During the continuous flow process in the system, rapid cells lysis with PCR-amplifiable genomic DNA were achieved within 20 minutes.     

Analysis of DNA sequences using a single chemical cleavage procedure

A novel approach to sequence analysis of end-labeled, defined DNA fragments, using a single chemical cleavage procedure and electrophoretic separation in a single lane, has been developed. Prolonged treatment with hot aqueous piperidine results in partial cleavage of the DNA at all positions; the relative propensity for this cleavage is different for the various bases in the DNA. The hydrolysate is resolved on a DNA sequencing gel, and the distribution of radioactivity in the electrophoretic lane is analyzed (a) in terms of differential peak heights of the radioactive bands and (b) in terms of the spacings between successive bands. Simultaneous application of these two base-characteristic criteria allows the deduction of the nucleotide sequence with an accuracy approaching that of the established four-lane methods of DNA sequencing.     

Chemical and biochemical postlabeling methods for singling out specific oxidative DNA lesions.

A survey of the main available chemical and biochemical postlabeling assays for measuring oxidative DNA damage is reported. Two main approaches, radio and fluorescent postlabeling, have been used in order to reach a high level of sensitivity of detection. This is required for the measurement of DNA damage within cells and tissues upon exposure to agents of oxidative stress. Most of the methods are based on liquid chromatographic separation of defined DNA modifications following either acidic hydrolysis or enzymic digestion of DNA. In a subsequent step, the isolated base or sugar damages are either radiolabeled or made fluorescent by chemical or enzymatic reactions. Emphasis is placed on the recently developed high performance liquid chromatographic 32P-postlabeling assay, which allows the specific and sensitive measurement of various base damages including adenine N-1 oxide and 5-hydroxymethyluracil at the level of one modification per 10(7) normal bases in a sample size of 1 microgram of DNA. Examples of application of radioactive postlabeling to the measurement of DNA base damage following exposure of human cells to oxidizing agents including hydrogen peroxide and UVA radiation are provided.     

Assembly of transcriptionally active chromatin in Xenopus oocytes requires specific DNA binding factors

Active minichromosomes assembled on injected 5S RNA gene clones are stable in Xenopus oocytes; endogenous 5S DNA specific factor(s) are required for their assembly. When somatic-type and oocyte-type 5S RNA gene clones are coinjected, the somatic genes are assembled into active minichromosomes, while most of the oocyte genes are assembled into inactive ones. The differential 5S RNA gene expression, which mimics that in somatic cells, appears to result from titration of 5S DNA specific factor(s) by the competing somatic 5S DNA, followed by histone mediated assembly of inactive chromatin on the oocyte 5S DNA. Stable minichromosomes are also assembled on a cloned histone H4 gene; again, intragenic DNA rearrangements affect the efficiency of assembly of active chromatin and differential gene expression occurs after coinjection of two or more H4 DNA constructs. We suggest that the H4 DNA molecules also compete for limiting quantities of specific DNA binding factor(s) required for the assembly of active H4 gene chromatin.     

In silico discovery and biophysical evaluation of novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase B

Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF).     

Endogenously generated DNA nucleobase modifications source,and significance as possible biomarkers of malignant transformation risk,and role in anticancer therapy

The DNA of all living cells undergoes continuous structural and chemical alteration, which may be derived from exogenous sources, or endogenous, metabolic pathways, such as cellular respiration, replication and DNA demethylation. It has been estimated that approximately 70,000 DNA lesions may be generated per day in a single cell, and this has been linked to a wide variety of diseases, including cancer. However, it is puzzling why potentially mutagenic DNA modifications, occurring at a similar level in different organs/tissue, may lead to organ/tissue specific cancers, or indeed non-malignant disease – what is the basis for this differential response? We suggest that it is perhaps the precise location of damage, within the genome, that is a key factor. Finally, we draw attention to the requirement for reliable methods for identification and quantification of DNA adducts/modifications, and stress the need for these assays to be fully validated. Once these prerequisites are satisfied, measurement of DNA modifications may be helpful as a clinical parameter for treatment monitoring, risk group identification and development of prevention strategies.     

Template-directed photoligation of oligodeoxyribonucleotides via 4-thiothymidine.

Non-enzymatic, template-directed ligation of oligonucleotides in aqueous solution has been of great interest because of its potential synthetic and biomedical utility and implications for the origin of life. Though there are many methods for template-directed chemical ligation of oligonucleotides, there are only three reported photochemical methods. In the first report, template-directed photoligation was effected by cyclobutane dimer formation between the 5'- and 3'-terminal thymidines of two oligonucleotides with >290 nm light, which also damages DNA itself. To make the photochemistry of native DNA more selective, we have replaced the thymidine at the 5'-end of one oligonucleotide with 4-thiothymidine (s4T) and show that it photoreacts at 366 nm with a T at the 3'-endof another oligonucleotide in the presence of a complementary template. When a single mismatch is introduced opposite either the s4T or its adjoining T, the ligation efficiency drops by a factor of five or more. We also show that by linking the two ends of the oligonucleotides together, photoligation can be used to form circular DNA molecules and to 'photopadlock' circular DNA templates. Thus, s4T-mediated photo-ligation may have applications to phototriggered antisense-based or antigene-based genetic tools, diagnostic agents and drugs, especially for those situations in which chemical or enzyme-mediated ligation isundesirable or impossible, for example inside a cell.     

Single-base resolution analysis of DNA epigenome via high-throughput sequencing

Epigenetic changes caused by DNA methylation and histone modifications play important roles in the regulation of various cellular processes and development. Recent discoveries of 5-methylcytosine (5mC) oxidation derivatives including 5-hydroxymethylcytosine (5hmC), 5-formylcytsine (5fC) and 5-carboxycytosine (5caC) in mammalian genome further expand our understanding of the epigenetic regulation. Analysis of DNA modification patterns relies increasingly on sequencing-based profiling methods. A number of different approaches have been established to map the DNA epigenomes with single-base resolution, as represented by the bisulfite-based methods, such as classical bisulfite sequencing (BS-seq), TAB-seq (TET-assisted bisulfite sequencing), oxBS-seq (oxidative bisulfite sequencing) and etc. These methods have been used to generate base-resolution maps of 5mC and its oxidation derivatives in genomic samples. The focus of this review will be to discuss the chemical methodologies that have been developed to detect the cytosine derivatives in the genomic DNA.     

Radiation-induced DNA damage and its repair

Application of modern methods of organic chemistry and recombinant DNA technologies has provided new insights in the field of DNA radiation damage and its repair. An overview of the chemical nature of the lesions inflicted on DNA by ionizing radiation is presented. The structures of 29 different DNA modified base or sugar residues are shown in comprehensive formation schemes. A fraction of radiation-induced modified bases is spontaneously released from the DNA chain during irradiation. Another part remains attached to the DNA chain backbone and for its characterization mild formic acid or enzymatic hydrolysis have been used. Starting from the chemical formulae of the altered base residues, the specific repair enzymes and their modes of action are discussed. Various glycosylases and endonucleases have been purified to homogeneity, and in some cases the gene which encodes the protein cloned. Using methods derived from Maxam and Gilbert sequencing procedures and DNA fragment 32P-labelled at one end, it has been shown that the alkali-labile sites in DNA induced by radiation are strongly dependent on the DNA base sequence. Enzymatic methods have been used to analyse the DNA base defects produced by gamma-irradiation of cells under in vivo conditions. Structures of modified bases were the same as those observed when DNA was irradiated in aqueous solution.     

Extraction of high molecular weight DNA from microbial mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.     

What' New: More advances in DNA sequencing technology

Since their introduction about ten years ago the rapid methods for sequencing DNA based either on selective chemical degradation¹ or primed enzymatic synthesis² have been subject to a number of modifications and improvements.^{3, 4} Two recently published papers describe further advances in these technologies: a method for obtaining information about DNA sequences directly from uncloned mammalian genomic DNA⁵ and a possible first step towards the automation of DNA sequencing⁶.     

Schistosoma mansoni: analysis of cercarial transformation methods

Four methods of transforming cercariae to schistosomulae in vitro in ELAC buffer (pH 7.2, 37 C, 0-6 hr incubation) were compared in relation to biochemical and ultrastructural characteristics. The transformation methods used were chemical (3 mM linoleate), mechanical (centrifuge/vortex), mechanical/chemical, and heat (incubation at 37 C). Ultrastructural characteristics examined were based on the presence or absence of glycocalyx, heptalaminate membrane, cyton granules, and nuclear condition. Two EM fixation methods were used. Biochemical parameters assayed were loss of water tolerance (uptake of trypan blue dye), eicosanoid biosynthesis (PGE, LTB4, and 5-HETE), protein synthesis (leucine uptake), RNA synthesis (uracil and orotic acid uptake), and DNA synthesis (thymidine uptake). EM characteristics were remarkably similar for all transformation methods except heat incubation, with transformed cercariae evidencing the characteristics of schistosomulae (cyton granule migration, absence of glycocalyx and heptalaminate membrane); however, euchromatic nuclei could not be demonstrated using in vivo or in vitro transformation methods. Despite the ultrastructural similarities between transformation methods, biochemical data demonstrated that the resultant organisms were quite different. The chemical transformation method gave the highest rate of loss of water tolerance and eicosanoid production. RNA and protein synthesis were not correlated to ultrastructural changes and were highest in those organisms undergoing mechanical transformation methods, significantly higher than in those cercariae transformed by the chemical method. DNA synthesis was not demonstrated using any transformation method, although thymidine uptake did occur. Our data indicate substantial biochemical differences exist between morphologically similar organisms. Thus, experiments using any type of artificially transformed schistosomule must be interpreted with caution until additional biochemical and physiological studies on cercarial transformation are undertaken.     

Chemical reactions in double-stranded nucleic acids. The nature of the bond formed during chemical ligation using a cis-diol group

Chemical and enzymatic ligation between the 5'-terminal phosphate of one oligonucleotide and the 3'-terminal 2',3'-cis-diol group of the other oligonucleotide on a complementary template was studied. Carbodiimide, imidazolide and N-hydroxybenzotriazole ester methods were used for chemical activation of the phosphate group, and T4 DNA ligase for enzymatic ligation. All the chemical activation methods produced 3',5'- and 2',5'-phosphodiester bonds (40-45 and 55-60%, resp.), whereas enzymatic ligation gave the product only with 3',5'-phosphodiester bond.     

DNA display II. Genetic manipulation of combinatorial chemistry libraries for small-molecule evolution

Biological in vitro selection techniques, such as RNA aptamer methods and mRNA display, have proven to be powerful approaches for engineering molecules with novel functions. These techniques are based on iterative amplification of biopolymer libraries, interposed by selection for a desired functional property. Rare, promising compounds are enriched over multiple generations of a constantly replicating molecular population, and subsequently identified. The restriction of such methods to DNA, RNA, and polypeptides precludes their use for small-molecule discovery. To overcome this limitation, we have directed the synthesis of combinatorial chemistry libraries with DNA "genes," making possible iterative amplification of a nonbiological molecular species. By differential hybridization during the course of a traditional split-and-pool combinatorial synthesis, the DNA sequence of each gene is read out and translated into a unique small-molecule structure. This "chemical translation" provides practical access to synthetic compound populations 1 million-fold more complex than state-of-the-art combinatorial libraries. We carried out an in vitro selection experiment (iterated chemical translation, selection, and amplification) on a library of 10(6) nonnatural peptides. The library converged over three generations to a high-affinity protein ligand. The ability to genetically encode diverse classes of synthetic transformations enables the in vitro selection and potential evolution of an essentially limitless collection of compound families, opening new avenues to drug discovery, catalyst design, and the development of a materials science "biology."     

Hydration effects on the duplex stability of phosphoramidate DNA-RNA oligomers.

Recent studies on uniformly modified oligonucleotides containing 3'-NHP(O)(O-)O-5'internucleoside linkages (3'amidate) and alternatively modified oligonucleotides containing 3'-O(O-)(O)PNH-5'internucleoside linkages (5'amidate) have shown that 3'amidate duplexes, formed with DNA or RNA complementary strands, are more stable in water than those of the corresponding phosphodiesters. In contrast, 5'amidates do not form duplexes at all. There is no steric reason that the 5'amidate duplex should not form. We demonstrate that these differences arise from differential solvation of the sugar-phosphate backbones. By molecular dynamics calculations on models of 10mer single-stranded DNA and double-stranded DNA-RNA molecules, both with and without the phosphoramidate backbone modifications, we show that the single-stranded 3'amidate and 5'amidate backbones are equally well solvated, but the 5'amidate backbone is not adequately solvated in an A-form duplex. These results are supported by quantum chemical free energy of solvation calculations which show that the 3'amidate backbone is favored relative to the 5'amidate backbone.