DamID

DAMiD (english DNA adenine methyltransferase identification) is a biochemical method for the determination of protein-DNA interactions in eukaryotes.

Principle

The DNA sequences which bind to the proteins are detected by means of a fusion protein of said DNA -binding protein and a DNA methyltransferase (DAM). After binding of the DNA binding protein methylation of adenosine residues in adjacent GATC sequences occurs by the attached DNA methyltransferase to N6 Methyladeninresten which would otherwise not occur in eukaryotes. Through the identification of the methylated adenosines the DNA sequences of the binding can be determined. The partially methylated DNA. Using the restriction endonuclease DpnI, which cuts DNA at methylated GATC sequences The ends of the DNA fragments are ligated with oligonucleotides. This labeled DNA is amplified in a polymerase chain reaction with primers, whose sequences are respectively complementary to the attached oligonucleotides. This means that only sequences are amplified adjacent to a DNA methylation.

Alternative methods are, for example, EMSA, ChIP, ChIP -on-chip or ChIP -Seq. In comparison to the chip methods not an antibody is required, it can not be checked participation post-translational modifications to the DNA binding by the protein. Furthermore, since only DNA sequences are detected, which was the Dam, and no protein-DNA interaction is directly detected, defective results with DNA clamp - containing proteins such as RNA polymerase, which have no fixed position on the DNA but slide along the DNA. It is due to the PCR reproduced only DNA sequences between two GATC sequences. The average distance between two consecutive GATC sequences is about 205 bases in Drosophila ( FlyBase release 5), 260 in mice ( MM9 ) and 264 in human ( HG19 ). Transfected plasmids should be produced coli strains in Dam -negative E., as lead bacterial Dam -methylated sequences to false positive results.