End labeling procedures

There are two ways to label a DNA molecular; by the ends or all along the molecule. End labeling can be performed at the 3′- or 5′-end. Labeling at the 3′ end is performed by filling 3′-end recessed ends with a mixture or labeled and unlabeled dNTPs using Klenow or T4 DNA polymerases. Both reactions are template dependent. Terminal deoxynucleotide transferase incorporates dNTPs at the 3′ end of any kind of DNA molecule or RNA. Labels incorporated at the 3′-end of the DNA molecule prevent any further extension or ligation to any other molecule, but this can be overcome by labeling the 5′-end of the desired DNA molecule. 5′-end labeling is performed by enzymatic methods (T4 polynucleotide kinase exchange and forward reactions), by chemical modification of sensitized oligonucleotides with phosphoroamidite, or by combined methods. Probe cleanup is recommended when high background problems occur, but caution should be taken not to damage the attached probe with harsh chemicals or by light exposure.