T7 DNA Polymerase

T7 DNA Polymerase



 The T7 DNA polymerase from T7 bacteriophage has 3’-5’ exonuclease and DNA polymerase activity but lacks 5’-3’ exonuclease domain, which is similar to T4 DNA polymerase. The processivity of this enzyme is quite good that is, the average length of DNA synthesized before the enzyme dissociates from the template, is considerably greater than for other enzymes. Thus, the average length of DNA synthesized by a single molecule of bacteriophage T7 polymerase is much greater than that of DNAs synthesized by other DNA polymerases. The binding and polymerization domain is occupied by the carboxy terminus while the potent 3’-5’exonuclease activity resides on the amino terminus.

The exonuclease activity is completed inactivated by incubating the enzyme with a reducing agent, molecular oxygen, and low concentrations of ferrous ions, for several days. Over 99% of the exonuclease activity is abolished without affecting the polymerization activity by these agents, which cause mutations and site specific modifications. The resulting chemically modified enzyme is marketed under the trade name Sequenase is ideal for determining the sequence of long tracts of DNA by the dideoxy mediated chain termination method. 

what is Terminal Deoxy Nucleotidyl Transferase?

what is Terminal Deoxy Nucleotidyl Transferase ?

Terminal transferase is an unusual DNA polymerase found only in prelymphocytes and in early staqes of lymphoid differentiation. Synthesis of single stranded tails at the 3’ ends of either single stranded DNA or double stranded DNA with protruding 3’ termini, by the enzyme Terminal Deoxy Nucleotidyl Transferase, is called tailing, can be used to generate protruding ends of defined sequence to facilitate cloning of fragments. It can be used to generate protruding ends of defined sequence, e.g poly A tails on the 3’ ends of the DNA insert and poly T tails on 3’ ends of the vector. Thus, the protruding ends of the DNA insert and vector will base pair under appropriate annealing conditions. Mg2+ cation is preferred when the nucleotide to be added is a purine while Co2+ is preferred for the addition of pyrimidines. The enzyme strongly prefers DNA with protruding 3’ terminus, although blunt or recessed 3’ termini are also used, but less efficiently, in buffers of low ionic strength with Co2+, Mg2+ or Mn2+ as bivalent cations. As many as thousands of deoxynucleotides can be incorporated using this enzyme on a template of DNA. Single nucleotide can be added to the 3’ termini of DNA if modified bases like dideoxynucleotides or cordycepin triphosphates are used instead of natural deoxynucleotide triphosphates.