110
reaction mixture containing MnCl 2 . Its half-life is 20 min at 95 °C and can synthesize DNA at 74 °C. It also lacks RNase H activity. This enzyme can be used to
amplify DNA from RNA templates containing GC-rich sequences or secondary
structures since it stays active at elevated temperatures used to denature such template RNA and increase primer hybridization specificity. Reverse transcription and
PCR amplification is possible with a single enzyme, eliminating the chance of
cross-contaminations that occurs frequently in two-steps RT-PCR. Tth pol has wide
applications in detection, quantization, and cloning of RNA due to its ability to catalyze both reverse transcription and DNA amplification. Tth DNA polymerase is apt
for use in RT-PCR of RNA up to 1000 bp, PCR amplification of single-stranded
DNA, primer extension reactions at elevated temperatures, and labeling of DNA
fragments with radionucleotides, digoxigenin, or biotin (Myers and Gelfand 1991).
4.7.4 C. therm Polymerase Klenow Fragment
The Klenow fragment of polymerase from Carboxydothermus hydrogenoformans
(C. therm.) has reverse transcriptase activity in the presence of Mg
2+
and is found to
be active up to 72 °C. It can catalyze the reverse transcription of mRNA, polyadenylated RNA, and viral RNA at high temperatures, which helps to reduce the effect
of secondary structures in mRNA and increases primer annealing specificity. In
addition to this, the enzyme also has 3′ → 5′ exonuclease proofreading activity.
4.8
Nucleases
Nucleases are enzymes that cleave the phosphodiester bonds in the nucleic acid
backbone. Deoxyribonucleases cleave DNA, while ribonucleases cleave
RNA. Nucleases are classified into exonucleases and endonucleases according to
their site of action. Exonucleases cleave end portion of nucleic acid molecules while
endonucleases cut internal sites in nucleic acids. Some enzymes show both exonuclease and endonuclease activities. Nucleases, especially restriction endonucleases
are of extreme importance in molecular biology for the manipulation of nucleic
acids for techniques like cloning.
Nucleases are ubiquitous in living organisms and are responsible for partial or
complete digestion of nucleic acids. They are involved in nucleic acid degradation,
senescence, apoptosis, replication, recombination, and repair.
4.8.1 Deoxyribonuclease
Deoxyribonucleases (DNase) catalyzes the hydrolysis of phosphodiester bonds in
DNA backbone, thus cleaving it. They are categorized into exo- and endonucleases
on the basis of their requirement for a free terminus for cleavage. Exonucleases can
cleave either 3′ or 5′ end of a DNA molecule, while endonucleases cleave internal
phosphodiester bonds either randomly or specifically depending on the enzyme.
G. Valsala and S. Sugathan
reaction mixture containing MnCl 2 . Its half-life is 20 min at 95 °C and can synthesize DNA at 74 °C. It also lacks RNase H activity. This enzyme can be used to
amplify DNA from RNA templates containing GC-rich sequences or secondary
structures since it stays active at elevated temperatures used to denature such template RNA and increase primer hybridization specificity. Reverse transcription and
PCR amplification is possible with a single enzyme, eliminating the chance of
cross-contaminations that occurs frequently in two-steps RT-PCR. Tth pol has wide
applications in detection, quantization, and cloning of RNA due to its ability to catalyze both reverse transcription and DNA amplification. Tth DNA polymerase is apt
for use in RT-PCR of RNA up to 1000 bp, PCR amplification of single-stranded
DNA, primer extension reactions at elevated temperatures, and labeling of DNA
fragments with radionucleotides, digoxigenin, or biotin (Myers and Gelfand 1991).
4.7.4 C. therm Polymerase Klenow Fragment
The Klenow fragment of polymerase from Carboxydothermus hydrogenoformans
(C. therm.) has reverse transcriptase activity in the presence of Mg
2+
and is found to
be active up to 72 °C. It can catalyze the reverse transcription of mRNA, polyadenylated RNA, and viral RNA at high temperatures, which helps to reduce the effect
of secondary structures in mRNA and increases primer annealing specificity. In
addition to this, the enzyme also has 3′ → 5′ exonuclease proofreading activity.
4.8
Nucleases
Nucleases are enzymes that cleave the phosphodiester bonds in the nucleic acid
backbone. Deoxyribonucleases cleave DNA, while ribonucleases cleave
RNA. Nucleases are classified into exonucleases and endonucleases according to
their site of action. Exonucleases cleave end portion of nucleic acid molecules while
endonucleases cut internal sites in nucleic acids. Some enzymes show both exonuclease and endonuclease activities. Nucleases, especially restriction endonucleases
are of extreme importance in molecular biology for the manipulation of nucleic
acids for techniques like cloning.
Nucleases are ubiquitous in living organisms and are responsible for partial or
complete digestion of nucleic acids. They are involved in nucleic acid degradation,
senescence, apoptosis, replication, recombination, and repair.
4.8.1 Deoxyribonuclease
Deoxyribonucleases (DNase) catalyzes the hydrolysis of phosphodiester bonds in
DNA backbone, thus cleaving it. They are categorized into exo- and endonucleases
on the basis of their requirement for a free terminus for cleavage. Exonucleases can
cleave either 3′ or 5′ end of a DNA molecule, while endonucleases cleave internal
phosphodiester bonds either randomly or specifically depending on the enzyme.
G. Valsala and S. Sugathan