Description

Yeast Poly(A) Polymerase uses ATP as a substrate for template-independent addition of AMP from ATP to the 3′ hydroxyl termini of RNA molecules. 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. Yeast Poly(A) Polymerase is recommended over T4 RNA Ligase for 3'-end labeling of long RNA molecules.

Applications

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Addition of a poly(A) tail to RNA synthesized in vitro for increasing RNA stability and translation efficiency prior to transferring into eukaryotic cells

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cDNA library construction using a primer containing poly(dT) on its 3′ end.

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Synthesis of polyadenylated RNA for nucleic acid amplification

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Source

A recombinant E. coli strain carrying yeast (Saccharomyces cerevisiae) Poly(A) Polymerase gene

Unit Definition

1 unit is defined as the amount of enzyme that will incorporate 1 nmol of ATP into acid-insoluble material in 10 minutes at 37°C.

Components

· Yeast Poly(A) Polymerase: 20 mM Tris-HCl, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, 50% Glycerol, 0.1% Triton® X-100, pH 7.5 @ 25°C

· 5X Reaction Buffer:  100 mM Tris-HCl, pH 7.0, 3.0 mM MnCl2, 50 mM MgCl₂,  0.5 mM EDTA, 5 mM DTT

Quality Control

The absence of endonuclease, nicking activity, exonuclease and RNAse activity is confirmed by appropriate quality tests.

Storage Condition

-20 °C