Biotechnology Index Glossary
Terminal transferase catalyzes the addition of nucleotides to the 3' terminus of DNA. Interestingly, it works on single-stranded DNA, including 3' overhangs of double-stranded DNA, and is thus an example of a DNA polymerase that does not require a primer. It can also add homopolymers of ribonucleotides to the 3' end of DNA.
The much preferred substrate for this enzyme is protruding 3' ends, but it will also, less efficiently, add nucleotides to blunt and 3'-recessed ends of DNA fragments. Cobalt is a necessary cofactor for activity of this enzyme.
Terminal transferase is useful for at least two procedures:
Labeling the 3' ends of DNA: Most commonly, the substrate for this reaction is a fragment of DNA generated by digestion with a restriction enzyme that leaves a 3' overhang, but oligodeoxynucleotides can also be used. When such DNA is incubated with tagged nucleotides and terminal transferase, a string of the tagged nucleotides will be added to the 3' overhang or to the 3' end of the oligonucleotide.
Adding complementary homopolymeric tails to DNA: This clever procedure was commonly used in the past to clone cDNAs into plasmid vectors, but has largely been replaced by other, much more efficient techniques. The principles of this technique are depicted in the figure below. Basically, terminal transferase is used to tail a linearized plasmid vector with G's and the cDNA with C's. When incubated together, the compementary G's and C's anneal to "insert" the cDNA into the vector, which is then transformed into E. coli.
Terminal transferase is a mammalian enzyme, expressed in lymphocytes. The enzyme purchased commercially is usually produced by expression of the bovine gene in E. coli.
Back to the index of Restriction Endonucleases and DNA Modifying Enzymes
Last updated on December 25, 1999
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