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A laboratory guide for in vivo studies of DNA methylation and protein/DNA interactions (Paperback, 1990)
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A laboratory guide for in vivo studies of DNA methylation and protein/DNA interactions (Paperback, 1990)
Series: Biomethods
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Total price: R1,535
Discovery Miles: 15 350
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A Safety Considerations Genomie sequencing involves a number oj
hazard- ous steps, such as high eurrent, high voltage, radioaetive
and highly toxie chemieals. It is, the- jore, absolutely essential
that the instruetions oj equipment manu/aeturers bejollowed and
that par- tieular attention is paid to the loeal and jederal safety
regulations. I Introduction 13 B Introduction Hypomethylation ofDNA
has been positively correlated with thc activation of many
eucaryotic genes. During the transition from inactive to active
genes changes in the protein/DNA interaction pattern occur. Tran-
scriptional activation of eucaryotic genes is mediated by specific
interac- tions oftransacting factors with their respective DNA
binding sites in Lhe control regions (promoters, enhancers) ofthe
genes. This process is ofLen accompanied by changes in local
chromatin strucLure, witnessed by the appearance of nuclease
hypersensitive sites, as weil as by changes in protein-DNA
interactions and, in the case of higher eucaryotes, alterations
ofthe cytosine methylation pattern. The sole available experimental
tech- nique that permits the study ofthe latter phenomena at single
nucleotide resolution is direct genomic sequencing/footprinting,
pioneered by Church and Gilbert (1984). This method combines the
chemical DNA- sequencing procedure of Maxam amI Gilbert (1980) with
thc detection 01' DNA sequences by electroblotting and indirect
end-Iabeling by hybridiza- tl0n. An alternative possibility is the
novel procedure (Saluz and . lost, 1989), using Taq polymerase. The
first steps 01' both meLhods are essen- tially the same: total
genomic DNA is digested wiLh a suilable restriction enzyme and the
resulting DNA fragments are chemically sequeneed.
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