Enhanced Diagnostic Tools
After sequencing the human genome scientists began to realize that just knowing the genetic information of an individual is not sufficient to understand its phenotype. The way a DNA sequence is translated into function does not directly depend on the sequence itself. It has now become clear that the interaction of genes with environmental factors plays a role as well. The new field of epigenetics attempts to integrate the different chemical interactions or languages that genomes and environment use to communicate with each other. Chemically, epigenetics can be described as structural adaptations of chromosomal regions that reflect silenced or activated states of genes in response of the epigenetic signaling network of a genome to environmental factors. DNA methylation, indicating a chemical epigenetic change or imprinting, is the most widely studied modification. The modification is found within CpG dinucleotides. DNA methylation is a biochemical process where a DNA base, usually cytosine, is enzymatically methylated at the 5-carbon position. This epigenetic modification is associated with gene regulation, and is of paramount importance to biological health and disease. Over the years a growing number of chemical modification at different amino-acid residues mostly located at histone tails have been identified as well. Examples are acetylation, methylation, phosphorylation, ubiquitination and sumoylation, and new types of modifications are still waiting to be discovered. Other epigenetically important features are factors like nuclear positioning, noncoding RNAs, and microRNAs, among others, which are also associated with gene regulation and chromatin structure. In biochemical and biological science the speed of discovery of novel findings goes hand in hand with the development of new techniques and/or scientific instrumentation. The past few years have witnessed the introduction of a whole host of new techniques that allow for the characterization of the epigenome. Specifically, the number of studies that attempt to characterize the epigenome of normal or altered genetic states has grown exponentially. The following tables outline a list of techniques and methods as well as current microarray platforms that have been developed for the study of the epigenome and their applications in biology and medicine.
CHROMATIN IMMUNOPRECIPITAION is a technique were intact nuclei are gently fixed to maintain the physical relationship of DNA-binding molecules to genomic DNA. The chromatin (DNA plus bound molecules) is sheared to small fragments and incubated with an antibody that selectively immuno-precipitates one of the bound molecules. The binding sites of the molecule (usually a protein) of interest become apparent from their enrichment in the immune-precipitated fraction of the genome.
Legend: TLC, thin-layer chromatography; RP-HPLC, reverse phase high performance liquid chromatography; Anti-5mC, anti-5-methylcytosine; RLGS, restriction landmark genome scanning; AP-PCR, arbitrarily primed polymerase chain reaction; AIMS, amplification of intermethylated sites.
Legend: MS-PCR, methylation-specific polymerase chain reaction; MS-SNuPE, methylation-specific single nucleotide primer extension; MS-SSCA, methylation- specific single-strand conformation analysis; MS-HRM, methylation-specific high resolution melting; CGI, CpGislandmicroarray; SMRT,submegabase resolution tiling array; MeDIP,methylated DNA immunoprecipitation; MeCIP,methyl-CpG immunoprecipitation; Oligonucleotides, Whole genome oligonucleotide array.
1 Sequencing of immunoprecipitated anti-5mC DNA
2 Methyl-binding protein precipitated sequencing.
3 Methyl-sensitive restriction enzyme sequencing.
4 Modified methylation-specific digital karyotyping.
Harrison & Parle-McDermott published a paper in 2011 describing the speed in the development of new techniques to study DNA methylation. The following figure illustrates this quite nicely.
Time line of DNA methylation analysis. Early techniques used in the 1980s allowed to measure the amount of 5-methylcytosine within a particular genome. Since then a variety of methods have been developed that allow for a more detailed study of the epigenome. These new type of methods or assays include the use of methylation-sensitive restriction enzymes, immunoprecipitation, bisulfite sequencing, usually in combination with the polymer chain reaction (PCR), the use of microarrays, reversed-phase high-performance liquid-chromatography (RP-HPLC), methylation-sensitive single nucleotide primer extension (MS-SnuPe), combined bisulfate restriction analysis (COBRA), arbitrarily primed PCR (APPCR), amplification of inter-methylated sites (AIMS), reduced representation bisulfite sequencing (RRBS), and finally next-generation sequencing, to name a few.
It is expected that next-generation sequencing approaches for DNA methylation analysis will dominate for a while. Newer sequencing technologies, such as single-molecule real-time sequencing (SMRT) are needed to directly detect all known DNA methylation reactions without the need for bisulfate treatment. As pointed out by Harrison and Parle-McDermott the major developments in the methodologies for profiling and fingerprinting the human methylome have followed a clear progression toward innovative sequencing techniques that allow for single-pair resolution. It is expected that as the technologies improve the cost of genome-wide sequencing will decrease. This will result in new waves of data and the need for better bioinformatics tools to allow for the accurate analysis of vast datasets in the coming years.
Weixing Feng, Zengchao Dong, Bo He and Kejun Wang; Analysis method of epigenetic DNA methylation to dynamically investigate the functional activity of transcription factors in gene expression. BMC Genomics 2012, 13:532 doi:10.1186/1471-2164-13-532.
Alan Harrison and Anne Parle-McDermott; DNA Methylation: A Timeline of Methods and Applications Front Genet. 2011; 2: 74.)
Lanlan Shena and Robert A. Waterland; Methods of DNA methylation analysis. Curr Opin Clin Nutr Metab Care 10:576–581. 2007 Lippincott Williams & Wilkins.
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