Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions for the development and function of living things. All known cellular life and some viruses contain DNA. The May 29, · Cell-free DNA in the blood provides a non-invasive diagnostic avenue for patients with cancer1. However, characteristics of the origins and molecular A microarray is a multiplex blogger.com purpose is to simultaneously detect the expression of thousands of genes from a sample (e.g. from a tissue). It is a two-dimensional array on a solid substrate—usually a glass slide or silicon thin-film cell—that assays (tests) large amounts of biological material using high-throughput screening miniaturized, multiplexed and parallel processing
Genome-wide cell-free DNA fragmentation in patients with cancer | Nature
DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoterDNA methylation typically acts to repress gene transcription. In mammals, DNA methylation is essential for normal development and is associated with a number of key processes including genomic imprintingX-chromosome inactivationrepression of transposable elementsagingand carcinogenesis.
As oftwo nucleobases have been found on which natural, enzymatic DNA methylation takes place: adenine and cytosine, dna microarray research paper. The modified bases are N 6 -methyladenine [1]5-methylcytosine [2] and N 4 -methylcytosine. Two of DNA's four bases, cytosine and adeninecan be methylated, dna microarray research paper. pombe but not N.
Methylation of cytosine to form 5-methylcytosine occurs at the same 5 position on the pyrimidine ring where the DNA base thymine 's methyl group is located; the same position distinguishes thymine from the analogous RNA base uracilwhich has no methyl group.
Spontaneous deamination of 5-methylcytosine converts it to thymine. This results in a T:G mismatch. Repair mechanisms then correct it back to the original C:G pair; alternatively, they may substitute A for G, turning the original C:G pair into a T:A pair, effectively changing a base and introducing a mutation.
This misincorporated dna microarray research paper will not be corrected during DNA replication as thymine is a DNA base. If the mismatch is not repaired and the cell enters the cell cycle the strand carrying the T will be complemented by an A in one of the daughter cells, such that the mutation becomes permanent.
The near-universal use of thymine exclusively in DNA and uracil exclusively in RNA may have evolved as an error-control mechanism, to facilitate the removal of uracils generated by the spontaneous deamination of cytosine. In plants and other organisms, DNA methylation is found in three different sequence contexts: CG or CpG dna microarray research paper, CHG or CHH where H correspond to A, T or C, dna microarray research paper. In mammals however, DNA methylation is almost exclusively found in CpG dinucleotides, with the cytosines on both strands being usually methylated.
Non-CpG methylation can however be observed in embryonic stem cells[13] [14] [15] and has also dna microarray research paper indicated in dna microarray research paper development.
The DNA methylation landscape of vertebrates is very particular compared to other organisms. High CpG methylation in mammalian genomes has an evolutionary cost because it dna microarray research paper the frequency of spontaneous mutations.
Loss of amino-groups occurs with a high frequency for cytosines, dna microarray research paper, with different consequences depending on their methylation. In mammals, the only exception for this global CpG depletion resides in a specific category of GC- and CpG-rich sequences termed CpG islands that are generally unmethylated and therefore retained the expected CpG content. DNA methylation was probably present at some extent in very early eukaryote ancestors.
In virtually every organism analyzed, methylation in promoter regions correlates negatively with gene expression. DNA methylation may affect the transcription of genes in two ways. First, the methylation of DNA itself may physically impede the binding of transcriptional proteins to the gene, [31] and second, and likely more important, methylated DNA may be bound by proteins known as methyl-CpG-binding domain proteins MBDs.
MBD proteins then recruit additional proteins to the locus, such as histone deacetylases and other chromatin remodeling proteins that can modify histonesthereby forming compact, inactive chromatin, termed heterochromatin.
This link dna microarray research paper DNA methylation and chromatin structure is very important. In particular, loss of methyl-CpG-binding protein 2 MeCP2 has been implicated in Rett syndrome ; and methyl-CpG-binding domain protein 2 MBD2 mediates the transcriptional silencing of hypermethylated genes in "cancer".
DNA methylation is a powerful transcriptional repressor, at least in CpG dense contexts. Transcriptional repression of protein-coding genes appears essentially limited to very specific classes of genes that need to be silent permanently and in almost all tissues.
While DNA methylation does not have the flexibility required for the fine-tuning of gene regulation, its stability is perfect to ensure the permanent silencing of transposable elements. DNA methylation of transposable elements has been known to be related to genome expansion. However, the evolutionary driver for genome expansion remains unknown. There is a clear correlation between the size of the genome and CpG, suggesting that the DNA methylation of transposable elements led to a noticeable increase in the mass of DNA.
A function that appears even more conserved than transposon silencing is dna microarray research paper correlated with gene expression. In almost all species where DNA methylation is present, DNA methylation is especially enriched in the body of highly transcribed genes.
A body of evidence suggests that it could regulate splicing [36] and suppress the activity of intragenic transcriptional units cryptic promoters or transposable elements. In yeast and mammals, H3K36 methylation is highly enriched in the body of highly transcribed genes.
In yeast at least, H3K36me3 recruits enzymes such as histone deacetylases to condense chromatin and prevent the activation of cryptic start sites. DNA methylation patterns are largely erased and then re-established between generations in mammals.
Almost all of the methylations from the parents are erased, dna microarray research paper, first during gametogenesisand again in early embryogenesiswith demethylation and remethylation occurring each time.
Demethylation in early embryogenesis occurs in the preimplantation period in two stages — initially in the zygotethen during the first few embryonic replication cycles of morula and blastula. A wave of methylation then takes place during the implantation stage of the embryo, with CpG islands protected from methylation. This results in global repression and allows housekeeping genes to be expressed in all cells. In the post-implantation stage, methylation patterns are stage- and tissue-specific, with changes that would define each individual cell type lasting stably over a long period.
In particular, DNA methylation appears critical for the maintenance of mono-allelic silencing in the context of genomic imprinting and X chromosome inactivation.
During embryonic development, few genes change their methylation status, at the important exception of many genes specifically expressed in the germline. By contrast, DNA methylation is dispensable in undifferentiated cell types, such as the inner cell mass of the blastocyst, primordial germ cells or embryonic stem cells. Since DNA methylation appears to directly regulate only a limited number of genes, how precisely DNA methylation absence causes the death of differentiated cells remain an open question.
Due to the phenomenon of genomic imprintingmaternal and paternal genomes are differentially marked and must be properly reprogrammed every time dna microarray research paper pass through the germline.
Therefore, dna microarray research paper, during gametogenesisprimordial germ cells must have their original biparental DNA methylation patterns erased and re-established based on the sex of the transmitting parent. After fertilization, the paternal and maternal genomes are once again demethylated and remethylated except for differentially methylated regions associated with imprinted genes.
This reprogramming is likely required for totipotency of the newly formed embryo and erasure of acquired epigenetic changes. In many disease processes, such as cancergene promoter CpG islands acquire abnormal hypermethylation, which results in transcriptional silencing that can be inherited by daughter cells following cell division. Hypomethylation, in general, arises earlier and is linked to chromosomal instability and loss of imprinting, whereas hypermethylation is associated with promoters and can arise secondary to gene oncogene suppressor silencing, but might be a target for epigenetic therapy.
Global hypomethylation has also been implicated in the development and progression of cancer through different mechanisms. Generally, in progression to cancer, hundreds of genes are silenced or activated, dna microarray research paper.
Although silencing of some genes in cancers occurs by mutation, a large proportion of carcinogenic gene silencing is a result of altered DNA methylation see DNA methylation in cancer. DNA methylation causing silencing in cancer typically occurs at multiple CpG sites in the CpG islands that are present in the promoters of protein coding genes. Altered expressions of microRNAs also silence or activate many genes in progression to dna microarray research paper see microRNAs in cancer.
Silencing of DNA repair genes through methylation of CpG islands in their promoters appears to be especially important in progression to cancer see methylation of DNA repair genes in cancer.
Epigenetic modifications such as DNA methylation have been implicated in cardiovascular disease, including atherosclerosis. In animal models of atherosclerosis, vascular tissue, as well as blood cells such as mononuclear blood cells, exhibit global hypomethylation with gene-specific areas of hypermethylation. DNA methylation polymorphisms may be used as an early biomarker of atherosclerosis since they are present before lesions are observed, which may provide an early tool for detection and risk prevention.
Two of the cell types targeted for DNA methylation polymorphisms are monocytes and lymphocytes, which experience an overall hypomethylation, dna microarray research paper. One proposed mechanism behind this global hypomethylation is elevated homocysteine levels dna microarray research paper hyperhomocysteinemiaa known risk factor for cardiovascular disease.
High plasma levels of homocysteine inhibit DNA methyltransferases, which causes hypomethylation. Hypomethylation of DNA affects dna microarray research paper that alter smooth muscle cell proliferation, dna microarray research paper, cause endothelial cell dysfunction, and increase inflammatory mediators, all of which are critical in forming atherosclerotic lesions.
Another gene that experiences a change in methylation status in atherosclerosis is the monocarboxylate transporter MCT3dna microarray research paper produces a protein responsible for the transport of lactate and other ketone bodies out of many cell types, including vascular smooth muscle cells.
In atherosclerosis patients, there is an increase in methylation of the CpG islands in exon 2, which decreases MCT3 protein expression. The downregulation dna microarray research paper MCT3 impairs lactate transport and significantly increases smooth muscle cell proliferation, which further contributes to the atherosclerotic lesion.
An ex vivo experiment using the demethylating agent Decitabine 5-aza-2 -deoxycytidine was shown to induce MCT3 expression in a dose dependent manner, as all hypermethylated sites in the exon 2 CpG island became demethylated after treatment. This may serve as a novel therapeutic agent to treat atherosclerosis, although no human studies have been conducted thus far. In addition to atherosclerosis described above, dna microarray research paper epigenetic changes have been identified in the failing human heart.
This may vary by disease etiology. For example, in ischemic heart failure DNA methylation changes have been linked to changes in gene expression that may direct gene expression associated with the changes in heart metabolism known to occur.
diabetic cardiomyopathy and co-morbidities e. obesity must be explored to see how common these mechanisms are. Most strikingly, in failing human heart these changes in DNA methylation are associated with racial and socioeconomic status which further impact how gene expression is altered, [55] and may influence how the individual's heart failure should be treated. In humans and other mammals, DNA methylation levels can be used to accurately estimate the age of tissues and cell types, forming an accurate epigenetic clock.
A longitudinal study of twin children showed that, between the ages of 5 and 10, there was divergence of methylation patterns due to environmental rather than genetic influences. High intensity exercise has been shown to result in reduced DNA methylation in skeletal muscle, dna microarray research paper.
A study that investigated the methylome of B cells along their differentiation cycle, using whole-genome bisulfite sequencing WGBSshowed that there is a hypomethylation from the earliest stages to the most differentiated stages. The largest methylation difference is between the stages of germinal center B cells and memory B cells.
Furthermore, this study showed that there is a similarity between B cell tumors and long-lived B cells in their DNA methylation signatures. Two reviews summarize evidence that DNA methylation alterations in brain neurons are important in learning and memory. Twenty four hours after contextual fear conditioning, 9. The hippocampus is needed to form memories, but memories are not stored there. For such mice, at four weeks after contextual fear conditioning, substantial differential CpG methylations and demethylations occurred in cortical neurons during memory maintenance, and there were 1, differentially methylated genes in their anterior cingulate cortex.
In mammalian cells, DNA methylation occurs mainly at the C5 position of CpG dinucleotides and is carried out by two general classes of enzymatic activities — maintenance methylation and de novo methylation, dna microarray research paper. Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle. Without the DNA methyltransferase DNMTthe replication machinery itself would produce daughter strands that are unmethylated and, over time, would lead to passive demethylation.
DNMT1 is the proposed maintenance methyltransferase that is responsible for copying DNA methylation patterns to the daughter strands during DNA replication. Mouse models with both copies of DNMT1 deleted are embryonic lethal at approximately day 9, due to the requirement of DNMT1 activity for development in mammalian cells, dna microarray research paper.
It is thought that DNMT3a and DNMT3b are the de novo methyltransferases that set up DNA methylation patterns early in development. DNMT3L is a protein that is homologous to the other DNMT3s dna microarray research paper has no catalytic activity.
Instead, DNMT3L assists the de novo methyltransferases by increasing their ability to bind to DNA and stimulating their activity. Mice and rats have a third functional de novo methyltransferase enzyme named DNMT3C, which evolved as a paralog of Dnmt3b by tandem duplication in the common ancestral of Muroidea rodents.
DNA Microarray
, time: 2:54Deoxyribonucleic Acid (DNA) Fact Sheet
Sep 02, · Phosphoramidite-based oligonucleotide synthesis is a prevalent and cost-effective technique to generate single-stranded DNA microarrays in which custom DNA sequences are printed at desired locations on a solid surface (Kosuri et al., ; LeProust et al., ).These microarrays can subsequently be converted into double-stranded DNA (dsDNA) microarrays by primer annealing and Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions for the development and function of living things. All known cellular life and some viruses contain DNA. The May 29, · Cell-free DNA in the blood provides a non-invasive diagnostic avenue for patients with cancer1. However, characteristics of the origins and molecular
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