VPB 321: Animal Biotechnology (2+1)

DIFFERENT WAYS OF GENERATING PROBES

Labeling method	Probe
5’ end labeling	Oligo / DNA	Replacement of 5’ phosphate group with labeled (gamma) g -phosphate group of free nucleotide catalyzed by T4 polynucleotide kinase
3’ end labeling	Oligo / DNA	Tailing with labeled nucleotides using terminal transferase
Nick translation	DNA	Nicks introduced into dsDNA by Dnase I and free 3’ ends extended by DNA polymerase I using labeled nucleotides
Random priming	DNA	Short random primers annealed to denatured DNA and extended by DNA polymerase using labeled nucleotides. Higher specific activity probes than nick translation
Primer extension	DNA	As above but using a specific primer. Used to label PCR products during thermal cycling
In vitro transcription	RNA	Single strand RNA produced by in vitro transcription using labeled nucleotide