(5) Steps 3 and 4 are repeated multiple times. To avoid the inconvenience of having to add new DNA polymerase in each cycle, a special DNA polymerase that can withstand heating to 1000C is used.
The idea is that in each cycle of melting, annealing and DNA synthesis, the amount of the DNA segment is doubled. This gives an exponential increase in the amount of the specific DNA as the cycles proceed. After 10 cycles the DNA is amplified 103 fold and after 20 cycles the DNA will be amplified 106 fold. Usually amplification is continued until all of the nucleotide precursors are incorporated into synthesized DNA.
Lecture 17. epigenetics as a way to control gene expression
Epigenetics refers to the study of heritable changes in gene expression that occur without a change in DNA sequence. Research has shown that epigenetic mechanisms provide an "extra" layer of transcriptional control that regulates how genes are expressed. These mechanisms are critical components in the normal development and growth of cells. Epigenetic abnormalities have been found to be causative factors in cancer, genetic disorders and pediatric syndromes as well as contributing factors in autoimmune diseases and aging. This lecture note introduces the basic principles of epigenetic mechanisms and their contribution to human health as well as the clinical consequences of epigenetic errors; also the use of epigenetic pathways in new approaches to diagnosis and targeted treatments across the clinical spectrum.
This new field will have an enormous impact on medicine, specifically on the study of heritable changes in gene function that do not change the DNA sequence but, rather, provide an "extra" layer of transcriptional control that regulates how genes are expressed. This rapidly evolving field offers exciting new opportunities for the diagnosis and treatment of complex clinical disorders. Basic principles of epigenetics are DNA methylation and histone modifications.
Dna methylation and histone modifications
(A) Schematic of epigenetic modifications. Strands of DNA are wrapped around histone octamers, forming nucleosomes, which to be organized into chromatin, the building block of a chromosome. Reversible and site-specific histone modifications occur at multiple sites through acetylation, methylation and phosphorylation. DNA methylation occurs at 5-position of cytosine residues in a reaction catalyzed by DNA methyltransferases (DNMTs). Together, these modifications provide a unique epigenetic signature that regulates chromatin organization and gene expression. (B) Schematic of the reversible changes in chromatin organization that influence gene expression: genes are expressed (switched on) when the chromatin is open (active), and they are inactivated (switched off) when the chromatin is condensed (silent). White circles = unmethylated cytosines; red circles = methylated cytosines.
Clinical consequences of epigenetic errors
Epigenetic mechanisms regulate DNA accessibility throughout a person's lifetime. Immediately following fertilization, the paternal genome undergoes rapid DNA demethylation and histone modifications.
27 The maternal genome is demethylated gradually, and eventually a new wave of embryonic methylation is initiated that establishes the blueprint for the tissues of the developing embryo. As a result, each cell has its own epigenetic pattern that must be carefully maintained to regulate proper gene expression. Perturbations in these carefully arranged patterns of DNA methylation and histone modifications can lead to congenital disorders and multisystem pediatric syndromes or predispose people to acquired disease states such as sporadic cancers and neurodegenerative disorders.
Aging
Both increases and decreases in DNA methylation are associated with the aging process, and evidence is accumulating that age-dependent methylation changes are involved in the development of neurologic disorders, autoimmunity and cancer in elderly people.
88 Methylation changes that occur in an age-related manner may include the inactivation of cancer-related genes. In some tissues, levels of methylated cytosines decrease in aging cells, and this demethylation may promote chromosomal instability and rearrangements, which increases the risk of neoplasia.
88 In other tissues, such as the intestinal crypts, increased global hypermethylation may be the predisposing event that accounts for the increased risk of colon cancer with advancing age.
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Cancer and epigenetic therapies
Cancer is a multistep process in which genetic and epigenetic errors accumulate and transform a normal cell into an invasive or metastatic tumour cell. Altered DNA methylation patterns change the expression of cancer-associated genes. DNA hypomethylation activates oncogenes and initiates chromosome instability,
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80 whereas DNA hypermethylation initiates silencing of tumour suppressor genes. The incidence of hypermethylation, particularly in sporadic cancers, varies with respect to the gene involved and the tumour type in which the event occurs.
To date, epigenetic therapies are few in number, but several are currently being studied in clinical trials or have been approved for specific cancer types.
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83 Nucleoside analogues such as azacitidine are incorporated into replicating DNA, inhibit methylation and reactivate previously silenced genes.
84 Azacitidine has been effective in phase I clinical trials in treating myelodysplastic syndrome and leukemias characterized by gene hypermethylation. The antisense oligonucleotide MG98 that downregulates DNMT1 is showing promising results in phase I clinical trials
86 and in targeting solid tumours and renal cell cancer (
www.methylgene.com/content.asp?node=14 [accessed 2005 Dec 22]). Similarly, small molecules such as valproic acid that downregulate HDACs are being used to induce growth arrest and tumour cell death. Combination epigenetic therapies (demethylating agents plus HDAC inhibitors) or epigenetic therapy followed by conventional chemotherapy (or immunotherapy) may be more effective since they reactivate silenced genes, including tumour suppressor genes, resensitize drug-resistant cells to standard therapies and act synergistically to kill cancer cells.
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The road ahead
Our increased knowledge of epigenetic mechanisms over the last 10 years is beginning to be translated into new approaches to molecular diagnosis and targeted treatments across the clinical spectrum. With the Human Genome Project completed, the Human Epigenome Project has been proposed and will generate genome-wide methylation maps.
106 By examining both healthy and diseased tissues, specific genomic regions will be identified that are involved in development, tissue-specific expression, environmental susceptibility and pathogenesis. Use of these epigenetic maps will lead to epigenetic therapies for complex disorders across the clinical spectrum.
