120
of only cohesive ends of DNA in a mixture containing both cohesive and blunt ends.
Applications include cloning of DNA fragments, joining of linkers or adapters, circularization of linear DNA, nick sealing, and in site-directed mutagenesis.
4.9.1.5 PBCV-1 DNA Ligase
PBCV-1 DNA ligase or Chlorella virus DNA Ligase, commercially known as
SplintR ligase is an ATP-dependent DNA ligase from Paramecium bursaria
Chlorella virus PBCV-1 (Ho et al. 1997). It catalyzes the ligation of two nicked
ssDNAs that are splinted by a complementary RNA strand, which has applications
in characterization of miRNAs and mRNAs. This enzyme is suitable for nextgeneration sequencing and molecular diagnostics. Its affinity for RNA-splinted
DNA substrates can be utilized for the detection of subnanomolar quantities of
unique RNA species within a complex mixture (Lohman et al. 2014).
4.9.1.6 Taq DNA Ligase
It is a thermostable DNA ligase isolated from Thermus aquaticus, which can ligate
cohesive ends in dsDNA with the help of NAD
+
as a cofactor. It can remain active
at temperatures as high as 45–65 °C and hence is used in techniques that require this
property (Housby et al. 2000). This enzyme is used in the detection of singlenucleotide polymorphism (SNP) using ligase detection reaction and ligase chain
reaction and mutagenesis by insertion of a phosphorylated oligonucleotide during
primer extension amplification.
4.9.1.7 Pfu DNA Ligase
Pfu DNA ligase is a thermostable ligase isolated from a hyperthermophilic marine
archaebacterium Pyrococcus furiosus. It catalyzes ligation of dsDNA at high temperatures ranging from 45 to 80 °C. Its optimum temperature for nick sealing is 70
°C. This enzyme has high ligation specificity and higher thermostability with a halflife of more than 1 h at 95 °C (Günther et al. 2002). It is used in ligase chain reactions (LCR) for higher reliability by permitting higher melting temperatures.
4.9.1.8 Tth DNA Ligase
Tth DNA ligase is a thermostable ligase isolated from Thermus thermophilus, which
can join dsDNA fragments and also repair single-stranded nicks in dsDNA. It catalyzes the formation of a phosphodiester bond between adjacent nucleotides hybridized to a complementary target DNA. This reaction will occur only if the two
nucleotides are perfectly paired to a complementary target DNA without any gaps.
This property can be applied for the detection of even a single-base substitution.
The high fidelity and thermostability of this enzyme makes it an excellent choice for
ligase chain reaction (LCR) and ligase detection reaction (LDR) to identify point
mutations associated with genetic disorders and pathogens (Luo et al. 1996).
G. Valsala and S. Sugathan
of only cohesive ends of DNA in a mixture containing both cohesive and blunt ends.
Applications include cloning of DNA fragments, joining of linkers or adapters, circularization of linear DNA, nick sealing, and in site-directed mutagenesis.
4.9.1.5 PBCV-1 DNA Ligase
PBCV-1 DNA ligase or Chlorella virus DNA Ligase, commercially known as
SplintR ligase is an ATP-dependent DNA ligase from Paramecium bursaria
Chlorella virus PBCV-1 (Ho et al. 1997). It catalyzes the ligation of two nicked
ssDNAs that are splinted by a complementary RNA strand, which has applications
in characterization of miRNAs and mRNAs. This enzyme is suitable for nextgeneration sequencing and molecular diagnostics. Its affinity for RNA-splinted
DNA substrates can be utilized for the detection of subnanomolar quantities of
unique RNA species within a complex mixture (Lohman et al. 2014).
4.9.1.6 Taq DNA Ligase
It is a thermostable DNA ligase isolated from Thermus aquaticus, which can ligate
cohesive ends in dsDNA with the help of NAD
+
as a cofactor. It can remain active
at temperatures as high as 45–65 °C and hence is used in techniques that require this
property (Housby et al. 2000). This enzyme is used in the detection of singlenucleotide polymorphism (SNP) using ligase detection reaction and ligase chain
reaction and mutagenesis by insertion of a phosphorylated oligonucleotide during
primer extension amplification.
4.9.1.7 Pfu DNA Ligase
Pfu DNA ligase is a thermostable ligase isolated from a hyperthermophilic marine
archaebacterium Pyrococcus furiosus. It catalyzes ligation of dsDNA at high temperatures ranging from 45 to 80 °C. Its optimum temperature for nick sealing is 70
°C. This enzyme has high ligation specificity and higher thermostability with a halflife of more than 1 h at 95 °C (Günther et al. 2002). It is used in ligase chain reactions (LCR) for higher reliability by permitting higher melting temperatures.
4.9.1.8 Tth DNA Ligase
Tth DNA ligase is a thermostable ligase isolated from Thermus thermophilus, which
can join dsDNA fragments and also repair single-stranded nicks in dsDNA. It catalyzes the formation of a phosphodiester bond between adjacent nucleotides hybridized to a complementary target DNA. This reaction will occur only if the two
nucleotides are perfectly paired to a complementary target DNA without any gaps.
This property can be applied for the detection of even a single-base substitution.
The high fidelity and thermostability of this enzyme makes it an excellent choice for
ligase chain reaction (LCR) and ligase detection reaction (LDR) to identify point
mutations associated with genetic disorders and pathogens (Luo et al. 1996).
G. Valsala and S. Sugathan