118
has high resistance to inactivation and denaturation by detergents, urea, or high concentrations of electrolyte, and is stable upon extended storage in the cold. F-Bal 31
nuclease is used in restriction site mapping, progressive deletion of sequences in
double-stranded DNA fragments from both termini, mapping B-Z DNA junctions,
and reducing the length of RNA molecules. The S-Bal 31 is used only for restriction
mapping and the controlled length reduction of linear duplex DNA.
4.8.4.5 Mung Bean Nuclease
This 39 kDa single-strand specific endonuclease can act on ssDNA and ssRNA, but
has no activity on dsDNA, dsRNA, or DNA-RNA hybrids under normal conditions.
However, high concentrations of enzyme can degrade double-stranded nucleic acids
when incubated for long duration. It is isolated from sprouts of mung bean Vigna
radiata. Zn
2+
is essential for the action of this enzyme while EDTA or SDS can
cause its irreversible inactivation (Johnson and Laskowski 1970). This enzyme is
ideal for the removal of 3′ and 5′ single-stranded overhangs of DNA and RNA molecules producing ligatable blunt ends, for the digestion of hairpin loops and in transcript mapping.
4.9
Polynucleotide Ligases
Polynucleotide ligases or ligases are enzymes that catalyze the joining of nucleic
acid molecules by the formation of phosphodiester bonds between them. Ligases
have been reported in various organisms including bacteria, phage-infected bacteria, yeasts, amphibians, and mammals, where they catalyze many important cellular
processes like DNA replication and repair of damaged DNA. Ligases are essential
for many techniques in recombinant DNA technology like cloning of cDNA or
genomic fragments for library construction, mapping, sequencing, or used as
probes. They are classified into DNA ligases and RNA ligases on the basis of their
nucleic acid specificity.
4.9.1 DNA Ligases
They catalyze the covalent joining of nicks in dsDNA by phosphodiester bond formation between a 3′-hydroxyl and a 5′-phosphate group, thereby joining the DNA
fragments (Lehman 1974). Inside a cell, they are involved in the linking of Okazaki
fragments during replication and in DNA repair. Bacterial DNA ligases require
NAD
+
as a cofactor while those in eukaryotes and viruses use ATP (Doherty and
Wigley 1999). The phosphodiester formation by all DNA ligases occurs in three
different catalytic steps. At first, ligase is activated through the formation of a covalent DNA ligase-AMP intermediate. Second step involves the transfer of AMP moiety to the 5′-phosphate terminus of nicked DNA strand and in the final step, the
DNA-AMP joins with the 3′-hydroxyl of the DNA break site, forming a
G. Valsala and S. Sugathan
has high resistance to inactivation and denaturation by detergents, urea, or high concentrations of electrolyte, and is stable upon extended storage in the cold. F-Bal 31
nuclease is used in restriction site mapping, progressive deletion of sequences in
double-stranded DNA fragments from both termini, mapping B-Z DNA junctions,
and reducing the length of RNA molecules. The S-Bal 31 is used only for restriction
mapping and the controlled length reduction of linear duplex DNA.
4.8.4.5 Mung Bean Nuclease
This 39 kDa single-strand specific endonuclease can act on ssDNA and ssRNA, but
has no activity on dsDNA, dsRNA, or DNA-RNA hybrids under normal conditions.
However, high concentrations of enzyme can degrade double-stranded nucleic acids
when incubated for long duration. It is isolated from sprouts of mung bean Vigna
radiata. Zn
2+
is essential for the action of this enzyme while EDTA or SDS can
cause its irreversible inactivation (Johnson and Laskowski 1970). This enzyme is
ideal for the removal of 3′ and 5′ single-stranded overhangs of DNA and RNA molecules producing ligatable blunt ends, for the digestion of hairpin loops and in transcript mapping.
4.9
Polynucleotide Ligases
Polynucleotide ligases or ligases are enzymes that catalyze the joining of nucleic
acid molecules by the formation of phosphodiester bonds between them. Ligases
have been reported in various organisms including bacteria, phage-infected bacteria, yeasts, amphibians, and mammals, where they catalyze many important cellular
processes like DNA replication and repair of damaged DNA. Ligases are essential
for many techniques in recombinant DNA technology like cloning of cDNA or
genomic fragments for library construction, mapping, sequencing, or used as
probes. They are classified into DNA ligases and RNA ligases on the basis of their
nucleic acid specificity.
4.9.1 DNA Ligases
They catalyze the covalent joining of nicks in dsDNA by phosphodiester bond formation between a 3′-hydroxyl and a 5′-phosphate group, thereby joining the DNA
fragments (Lehman 1974). Inside a cell, they are involved in the linking of Okazaki
fragments during replication and in DNA repair. Bacterial DNA ligases require
NAD
+
as a cofactor while those in eukaryotes and viruses use ATP (Doherty and
Wigley 1999). The phosphodiester formation by all DNA ligases occurs in three
different catalytic steps. At first, ligase is activated through the formation of a covalent DNA ligase-AMP intermediate. Second step involves the transfer of AMP moiety to the 5′-phosphate terminus of nicked DNA strand and in the final step, the
DNA-AMP joins with the 3′-hydroxyl of the DNA break site, forming a
G. Valsala and S. Sugathan