Poly(A) Polymerase catalyses the addition of adenosine residues onto the 3' ends of RNA. It can be used to add poly(A) tails to RNA in the first step of cloning. The reaction requires Mn2+ or Mg2+, ATP as substrate, and any RNA containing 3' hydroxyl termini as primer. Substitution of cordycepin-5'-triphosphate (3'-dATP) for ATP results in addition of a single 3'-dA residue to the ends of the RNA, a useful technique for labeling RNA at the 3' end.
In comparative studies, yeast poly(A) polymerase works more efficiently than E. coli poly(A) polymerase for RNA oligonucleotide-labeling and poly(A) tailing. Shorter incubation times are required for the yeast enzyme and it is found to label both long and short substrates equally well. Poly(A) Polymerase is recommended over T4 RNA Ligase for 3'-end labeling of long RNA molecules.
*Note: Various modified nucleotides can also be used to label the 3' end of RNA using Yeast Poly(A) Polymerase.
20 mM Tris-HCl (pH 8.0), 50 mM KCl, 0.5 mM DTT, 50% glycerol.
2 5mM Tris-HCl (pH 7.0), 40 mM KCl, 0.5 mM MnCl2, 0.05 mM EDTA, 0.5 mM DTT, 0.2 mg/mL BSA, 10% glycerol, 3.3 µM radiolabeled ATP, 0.5mM ATP, 6.5 µg poly(A) (∼100 bases), poly(A) polymerase. After incubation at 37°C for 10 min, acid insoluble radioactivity is determined.
One unit equals 15 pmol/min AMP incorporated into (riboA)15 at 37°C.
3' -end labeling of a ribonucleotide with cordycepin-5'-triphosphate.
Functionally Tested 5X Poly(A) Polymerase Reaction Buffer (1 mL included, PN 74226):
100 mM Tris-HCl, pH 7.0, 3.0 mM MnCl2, 0.1 mM EDTA, 1mM DTT, 500 µg/mL acetylated BSA, 50% glycerol.
1.Addition of poly(A) tails to RNA.
2.Labeling the 3' ends of RNA.
E. coli strain containing an overproducing clone of Yeast Poly(A) Polymerase.
For Research Use Only. Not for use in diagnostic procedures.