119
phosphodiester bond with the release of free AMP. DNA ligases are routinely used
in molecular biology for joining DNA fragments produced by restriction digestion,
for the addition of linkers or adaptors to DNA, or to repair nicks in DNA. They can
join both blunt and sticky ends of DNA. Many thermostable DNA ligases have been
isolated from thermophilic microbes, which have application in techniques like
ligase chain reaction (LCR) used for identifying single-base mutations.
4.9.1.1 E. coli DNA Ligase
It is a monomeric NAD
+
-dependent 77 kDa ligase enzyme that prefers sticky dsDNA
ends, but can also join blunt ends in the presence of Ficoll or polyethylene glycol.
E. coli DNA ligase can be used for reactions where double-stranded sticky end
DNA ligation is required along with prevention of blunt end ligation (Panasenko
et al. 1978). It has applications in molecular cloning of dsDNA with cohesive ends,
cDNA cloning of second strand cDNA synthesis product, and DNA cloning by
replacement synthesis where its inability to ligate RNA to DNA is an advantage.
4.9.1.2 T4 DNA Ligase
It is the most widely used DNA ligase in molecular biology. T4 DNA ligase is an
elongated monomeric enzyme of 68 kDa size. It was isolated from E. coli infected
with T4 bacteriophage. This enzyme requires Mg
2+
and ATP as cofactors for its
activity. It can be used for joining both blunt and sticky ends and can repair singlestranded nicks in dsDNA, dsRNA, or DNA-RNA hybrids. Ligation of blunt-ended
DNA is slower, but the rate can be increased by the addition of 150–200 mM NaCl
and low concentration of PEG. The enzyme also behaving as an AMP-dependent
endonuclease, producing nicked DNA which along with the ligase activity result in
complete relaxation of supercoiled DNA (Murray et al. 1979). T4 DNA ligase is
mainly used in the cloning of restriction digestion fragments and connecting synthetic linkers and adapters to DNA blunt ends.
4.9.1.3 T3 DNA Ligase
T3 DNA ligase, isolated from T3 bacteriophage is an ATP-dependent dsDNA ligase
that can ligate sticky ends and blunt ends and also repair nicks in dsDNA. As with
T4 DNA ligase, its efficiency in blunt-ended ligation is low, but can be enhanced
using PEG 6000. It has high tolerance for NaCl and KCl when compared to T4
DNA ligase. T3 DNA ligase is higher than five times more efficient than T4 DNA
ligase in ligation of sticky DNA ends. It is more phylogenetically similar to T7 DNA
ligase than T4 DNA ligase (Cai et al. 2004). T3 DNA ligase is used in DNA cloning,
joining of linkers or adapters, circularization of linear DNA and in nick sealing.
4.9.1.4 T7 DNA Ligase
It is a 41 kDa ATP-dependent dsDNA ligase isolated from E. coli infected with T7
bacteriophage. It catalyzes the ligation of sticky ends of duplex DNA and singlestranded nick sealing (Doherty et al. 1996). It cannot efficiently ligate blunt ends under
typical reaction conditions, but the addition of high concentrations of PEG 6000 can
improve its activity. T7 DNA ligase is used in techniques that require selective ligation
4 Enzymes as Molecular Tools
phosphodiester bond with the release of free AMP. DNA ligases are routinely used
in molecular biology for joining DNA fragments produced by restriction digestion,
for the addition of linkers or adaptors to DNA, or to repair nicks in DNA. They can
join both blunt and sticky ends of DNA. Many thermostable DNA ligases have been
isolated from thermophilic microbes, which have application in techniques like
ligase chain reaction (LCR) used for identifying single-base mutations.
4.9.1.1 E. coli DNA Ligase
It is a monomeric NAD
+
-dependent 77 kDa ligase enzyme that prefers sticky dsDNA
ends, but can also join blunt ends in the presence of Ficoll or polyethylene glycol.
E. coli DNA ligase can be used for reactions where double-stranded sticky end
DNA ligation is required along with prevention of blunt end ligation (Panasenko
et al. 1978). It has applications in molecular cloning of dsDNA with cohesive ends,
cDNA cloning of second strand cDNA synthesis product, and DNA cloning by
replacement synthesis where its inability to ligate RNA to DNA is an advantage.
4.9.1.2 T4 DNA Ligase
It is the most widely used DNA ligase in molecular biology. T4 DNA ligase is an
elongated monomeric enzyme of 68 kDa size. It was isolated from E. coli infected
with T4 bacteriophage. This enzyme requires Mg
2+
and ATP as cofactors for its
activity. It can be used for joining both blunt and sticky ends and can repair singlestranded nicks in dsDNA, dsRNA, or DNA-RNA hybrids. Ligation of blunt-ended
DNA is slower, but the rate can be increased by the addition of 150–200 mM NaCl
and low concentration of PEG. The enzyme also behaving as an AMP-dependent
endonuclease, producing nicked DNA which along with the ligase activity result in
complete relaxation of supercoiled DNA (Murray et al. 1979). T4 DNA ligase is
mainly used in the cloning of restriction digestion fragments and connecting synthetic linkers and adapters to DNA blunt ends.
4.9.1.3 T3 DNA Ligase
T3 DNA ligase, isolated from T3 bacteriophage is an ATP-dependent dsDNA ligase
that can ligate sticky ends and blunt ends and also repair nicks in dsDNA. As with
T4 DNA ligase, its efficiency in blunt-ended ligation is low, but can be enhanced
using PEG 6000. It has high tolerance for NaCl and KCl when compared to T4
DNA ligase. T3 DNA ligase is higher than five times more efficient than T4 DNA
ligase in ligation of sticky DNA ends. It is more phylogenetically similar to T7 DNA
ligase than T4 DNA ligase (Cai et al. 2004). T3 DNA ligase is used in DNA cloning,
joining of linkers or adapters, circularization of linear DNA and in nick sealing.
4.9.1.4 T7 DNA Ligase
It is a 41 kDa ATP-dependent dsDNA ligase isolated from E. coli infected with T7
bacteriophage. It catalyzes the ligation of sticky ends of duplex DNA and singlestranded nick sealing (Doherty et al. 1996). It cannot efficiently ligate blunt ends under
typical reaction conditions, but the addition of high concentrations of PEG 6000 can
improve its activity. T7 DNA ligase is used in techniques that require selective ligation
4 Enzymes as Molecular Tools