101
sequences of catalytic region (Burgers et al. 2001). They all share similar structural
configuration and have conserved regions in their catalytic site (Franklin et al. 2001;
Steitz 1998). DNA polymerases used in molecular biology belong to families A and
B. They are used for DNA manipulations like terminal or whole sequence labeling,
modification of DNA termini, production of double-stranded cDNA, DNA sequencing, extension of synthetic DNA sequences for gene synthesis, site-directed mutagenesis, and amplification of a particular portion of DNA for analysis.
DNA polymerase I of Escherichia coli was discovered by Arthur Kornberg in
1956 (Kornberg 1957). Sanger et al. (1977) used it for developing dideoxy sequencing of DNA. Kary Mullis in 1985 developed the PCR technique (Cetus Corporation,
California, USA) and was awarded the Nobel Prize in Chemistry in 1993 (Mullis
et al. 1986). Initially, PCR was carried out with the Klenow fragment of DNA polymerase I from E. coli, but being sensitive to the high temperature used for denaturation step, it had to be replenished after each cycle. This was resolved by the use of
a thermostable DNA polymerase from Thermus aquaticus, a thermophilic bacterium isolated from geysers in Yellowstone National Park, Montana, USA (Saiki
et al. 1988). Since then, polymerases from a number of thermophilic microorganisms like Pfu polymerase from Pyrococcus furiosus, Wind or Tli polymerase or
Vent polymerase from Thermococcus litoralis, and Tth polymerase from Thermus
thermophilus have been developed for use in molecular biology. Initially, the amplification capacity of PCR was limited to a short stretch of DNA of up to 10 kb size.
With the development of recombinant polymerases, it became possible to amplify
fragments up to 70 kb size (Blanco et al. 1989).
A wide selection of DNA polymerases with unique properties is currently available with different rates of extension and proofreading activity (Table 4.1). The
choice of polymerase is based on the technique used and the type and size of template. PCR technique has revolutionized medical and forensic science and is currently used for the detection of hereditary diseases, gene cloning, DNA fingerprinting,
parentage testing, and in the detection of infectious diseases like AIDS.
4.3
Terminal Deoxynucleotidyl Transferases
Terminal deoxyribonucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) is a template-independent DNA polymerase first identified
in calf thymus. It is expressed in mammalian lymphocytes and is currently manufactured commercially by the over expression of the bovine gene DNA nucleotidylexotransferase (DNTT) in E. coli. This enzyme catalyzes the incorporation of
deoxynucleotides to the 3′-OH termini of DNA without the help of a template. It
can efficiently act on 3′ protruding ends, while addition of nucleotides to blunt and
3′-recessed ends of DNA is of low efficiency. TdT lacks 3′ → 5′ and 5′ → 3′ exonuclease activities (Chang and Bollum 1986). Cobalt is an essential cofactor
required for the functioning of this enzyme.
4 Enzymes as Molecular Tools
sequences of catalytic region (Burgers et al. 2001). They all share similar structural
configuration and have conserved regions in their catalytic site (Franklin et al. 2001;
Steitz 1998). DNA polymerases used in molecular biology belong to families A and
B. They are used for DNA manipulations like terminal or whole sequence labeling,
modification of DNA termini, production of double-stranded cDNA, DNA sequencing, extension of synthetic DNA sequences for gene synthesis, site-directed mutagenesis, and amplification of a particular portion of DNA for analysis.
DNA polymerase I of Escherichia coli was discovered by Arthur Kornberg in
1956 (Kornberg 1957). Sanger et al. (1977) used it for developing dideoxy sequencing of DNA. Kary Mullis in 1985 developed the PCR technique (Cetus Corporation,
California, USA) and was awarded the Nobel Prize in Chemistry in 1993 (Mullis
et al. 1986). Initially, PCR was carried out with the Klenow fragment of DNA polymerase I from E. coli, but being sensitive to the high temperature used for denaturation step, it had to be replenished after each cycle. This was resolved by the use of
a thermostable DNA polymerase from Thermus aquaticus, a thermophilic bacterium isolated from geysers in Yellowstone National Park, Montana, USA (Saiki
et al. 1988). Since then, polymerases from a number of thermophilic microorganisms like Pfu polymerase from Pyrococcus furiosus, Wind or Tli polymerase or
Vent polymerase from Thermococcus litoralis, and Tth polymerase from Thermus
thermophilus have been developed for use in molecular biology. Initially, the amplification capacity of PCR was limited to a short stretch of DNA of up to 10 kb size.
With the development of recombinant polymerases, it became possible to amplify
fragments up to 70 kb size (Blanco et al. 1989).
A wide selection of DNA polymerases with unique properties is currently available with different rates of extension and proofreading activity (Table 4.1). The
choice of polymerase is based on the technique used and the type and size of template. PCR technique has revolutionized medical and forensic science and is currently used for the detection of hereditary diseases, gene cloning, DNA fingerprinting,
parentage testing, and in the detection of infectious diseases like AIDS.
4.3
Terminal Deoxynucleotidyl Transferases
Terminal deoxyribonucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) is a template-independent DNA polymerase first identified
in calf thymus. It is expressed in mammalian lymphocytes and is currently manufactured commercially by the over expression of the bovine gene DNA nucleotidylexotransferase (DNTT) in E. coli. This enzyme catalyzes the incorporation of
deoxynucleotides to the 3′-OH termini of DNA without the help of a template. It
can efficiently act on 3′ protruding ends, while addition of nucleotides to blunt and
3′-recessed ends of DNA is of low efficiency. TdT lacks 3′ → 5′ and 5′ → 3′ exonuclease activities (Chang and Bollum 1986). Cobalt is an essential cofactor
required for the functioning of this enzyme.
4 Enzymes as Molecular Tools