Reverse Transcriptase AMV
From Avian Myeloblastosis Virus
- Highly active aβ dimer
- Amplification of RNA
- No 5' to 3' exonuclease activity
- RNA-directed DNA polymerase
- DNA-dependent DNA polymerase
- RNase H and unwinding activity
AMV Reverse Transcriptase is an RNA-dependent DNA polymerase that catalyzes the polymerization of nucleotides using template DNA, RNA or RNA:DNA hybrids. It is widely used for first and second strand synthesis of complementary DNA (cDNA) from mRNA templates for cloning or for hybridization probes. Such synthesis requires a primer, commonly oligo(dT) bound to the 3'-poly(A) region of mRNA. Polymerization proceeds in the 5' to 3' direction with no 5' to 3' exonuclease activity. Under the right conditions, high yields of full length cDNA can be obtained using AMV reverse transcriptase.
AMV reverse transcriptase is one of the gene products of the RNA-genome of the avian myeloblastosis virus. The enzyme is composed of two polypeptides, α and β. The molecular weight of the α subunit is 68,000 Da and that of the β subunit 92,000 Da.
The α subunit is derived from the βsubunit by proteolysis and forms an αβ dimer which is the most active form of the enzyme. This mature αβ form exhibits several enzymatic activities: a RNA-directed DNA polymerase, a DNA-dependent DNA polymerase, an RNase H, and an unwinding activity.
Reverse Transcriptase is used chiefly to transcribe mRNA into cDNA which can then be cloned into vectors or amplified by PCR. It may also be used for filling in and labeling the 3'-termini of DNA with 5'-protruding ends. AMV Reverse Transcriptase can also be used for dideoxy DNA sequencing and RNA sequencing.
|Storage:||200 mM KPO4 pH 7.2, 2 mM DTT, 0.2% Triton X-100, 50% Glycerol, temperature -20°C.|
|Incubation buffer:||50mM Tris HCl pH 8.3, 40 mM KCl, 6 mM MgCl2.|
|Unit definition:||One unit incorporates 1 nmol of dTTP into an acid-insoluble product in 10 minutes at 37°C using polyA-oligo dT12-18 as a substrate.|
|Purity:||Free of contaminating endonuclease, exonuclease and ribonuclease.|
|Activity:||10-20 units/µL with a specific activity higher than 200,000 units per mg.|
|Functional testing:||When globin mRNA is incubated for 5 minutes at 37°C, at least 99% of cDNA products are full length.|
Includes 10X incubation buffer