Drawn illustration of the DamID concept.
DamID (DNA adenine methyltransferase identification) is a technique for detecting exactly where a chromatin protein binds to the DNA. The technique was invented in 2000 by Bas van Steensel of the Dutch Cancer Institute.
The basis of the art is to link to known chromatin proteins a subunit leaving a trace (methyl groups) on the portion of the DNA to which the protein binds. In this way, it is accurate to determine the binding points of the various chromatin proteins. At some points, DamID is more accurate than Chromatin Immuno precipitation. Technology
Escherichia coli possesses the DNA methyltransferase Dam that methylates only adenine bases. By incorporating the gene encoding Dam on the 3 'side of a chromatin protein, the protein receives a Dam subunit. When the Dam fusion protein binds to DNA, the Dam subunit adenines will methylate in a GATC sequence upstream and downstream of the binding site. The binding sites thereafter are easy to recognize because methylated adenines do not occur in eukaryotes.
The sites where adenines are methylated can be detected by digesting the treated DNA with the restriction enzyme Digest Dpnl, as it cuts only in methylated GATC. The fragments are then subjected to a Polymerase chain reaction, Southern blot and a DNA microarray.
A difficulty with this technique is that the fusion proteins do not always bind perfectly to their binding plates; This results in a large background of methylation. Also, some GATC sequences are more accessible to Dam than others. To improve the results, a number of control experiments have to be done. A negative check must be made that adds the same genome unbound Dam, the results of the negative check must be deducted from the results of the expense. A positive check should also be made, comparing the results of the experiment with a test where the binding sites are known. Pros and cons of ChIP
Benefits of DamID over ChIP is that no immunoglobulins are required for DamID. DamID requires much less cells to get good results.
One major drawback is that it takes hours to run a DamID while a ChIP takes a few minutes. At ChIP there is a noise in the labeling of 0.5-1 kilobases, at DamID it is 1-2 kilobases!
When comparing the results of ChIP and DamID, there are approximately the same results.
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