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DNA laddering (left) visualised in an agarose gel by ethidium bromide staining. A 1 kb marker (middle) and control DNA (right) are included.. DNA laddering is a feature that can be observed when DNA fragments, resulting from Apoptosis DNA fragmentation are visualized after separation by gel electrophoresis the first described in 1980 by Andrew Wyllie at the University Edinburgh medical school ...
The apoptotic DNA fragmentation is being used as a marker of apoptosis and for identification of apoptotic cells either via the DNA laddering assay, [2] the TUNEL assay, [3] [4] or the by detection of cells with fractional DNA content ("sub G 1 cells") on DNA content frequency histograms e.g. as in the Nicoletti assay.
A molecular-weight size marker, also referred to as a protein ladder, DNA ladder, or RNA ladder, is a set of standards that are used to identify the approximate size of a molecule run on a gel during electrophoresis, using the principle that molecular weight is inversely proportional to migration rate through a gel matrix.
Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis. In the original publication of 1977, [ 6 ] the formation of base-paired loops of ssDNA was a cause of serious difficulty in resolving bands at some locations.
When an electric field is applied, the DNA will begin to move through the gel, at a speed roughly inversely proportional to the length of the DNA molecule (shorter lengths of DNA travel faster) — this is the basis for size dependent separation in standard electrophoresis. In TGGE there is also a temperature gradient across the gel.
Once DNA-protein binding is determined in vitro, a number of algorithms can narrow the search for identification of the transcription factor. Consensus sequence oligonucleotides for the transcription factor of interest will be able to compete for the binding, eliminating the shifted band, and must be confirmed by supershift.
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The key to cloning a DNA fragment is to link it to a vector DNA molecule that can replicate within a host cell. After a single recombinant DNA molecule (composed of a vector plus an inserted DNA fragment) is introduced into a host cell, the inserted DNA can be replicated along with the vector, generating a large number of identical DNA ...