Random priming
As random priming (or random-primed oligo labeling ) in which is molecular biology , a technique for labeling of DNA , respectively. A marked opposite strand is synthesized on denatured (single-stranded) DNA with the aid of short primers as a starting point.
At the beginning, the DNA to be labeled is denatured. Then a mixture of oligonucleotides , usually hexaoligonucleotides, more rarely also somewhat longer ones, with a random sequence is added and these are attached to the opposite strand in a phase known as annealing . The fragment of DNA polymerase I from E. coli known as the Klenow fragment uses these primers to synthesize the opposite strand with the aid of labeled nucleotides. The Klenow fragment is used because it can synthesize from 5 'to 3' ( polymerase activity) and cut out nucleotides from 3 'to 5' ( exonuclease activity), but does not cut from 5 'to 3'. The Klenow fragment falls off the original after an average of 500 to 2000 bases.
As in nick translation, the DNA can be labeled by using radioactively labeled (e.g. [α- 32 P] dATP ) or nucleotides linked to a labeling molecule ( digoxygenin , biotin , fluorescent dye ) . Because of the short strands to be synthesized, this system is very simple and fast. Before use, the DNA molecules, e.g. B. by size exclusion chromatography, separated from the non-incorporated nucleotides.
literature
- AP Feinberg & B. Vogelstein (1983): A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. In: Anal. Biochem. Vol. 132, pp. 6-13. PMID 6312838