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zinc and magnesium ions and is inactivated by chelating agents (Mössner et al.
1980).
4.10.1.3 Shrimp Alkaline Phosphatase
It is a highly specific, heat-sensitive phosphatase enzyme isolated from arctic shrimp
(Pandalus borealis). Its catalytic action is similar to BAP and CIP, but being heat
sensitive, it can be irreversibly inactivated by heat treatment at 65 °C for 15mins. It
is used for 5′ dephosphorylation of DNA or RNA for subsequent labeling of the
ends, to prevent self-ligation, and for the inactivation of unused dNTPs from PCR
product prior to sequencing.
4.10.2 Acid Phosphatases
Acid phosphatases have optimal pH below 7.0. It is commercially produced from
wheat germ. Three isozymes of acid phosphatase have been isolated, namely EI,
EII, and EIII with similar molecular weights (55 kDa ± 5 kDa) and having optimum
pH’s 5.5, 4.5, and 4.0, respectively (Verjee 1969).
4.10.2.1 Tobacco Acid Pyrophosphatase
Tobacco acid pyrophosphatase (TAP) catalyzes the hydrolysis of various pyrophosphate bonds, including the triphosphate bridge of 5′-terminal methylated guanine
nucleotide cap of mRNA, generating a 5′-phosphate terminus which can be dephosphorylated with alkaline phosphatase, and labeled with T4 polynucleotide kinase
for radiolabeled probe production or ligated with oligonucleotide adaptors for
RACE. EpiCentre, the only manufacturer of TAP, has discontinued its production.
TAP was produced directly from tobacco callus tissue due to difficulties in making
a recombinant enzyme. Decapping Pyrophosphohydrolase from tebu-bio and CapClip™ Acid Pyrophosphatase from CellScript are the available replacements for
TAP of which the latter was found to have better catalytic activity.
4.10.3 T4 Polynucleotide Kinase
T4 polynucleotide kinase, isolated from T4 bacteriophage infected E. coli, is the
most widely used polynucleotide kinase in molecular biology. It is a homotetramer
of 33-kDa monomers encoded by pse T gene. T4 polynucleotide kinase has phosphatase activity at 3′ end and kinase activity at 5′ end. It catalyzes the transfer of a
phosphate group from ATP molecule to the 5′ end of double-stranded and singlestranded DNA or RNA or nucleoside 3′-monophosphates. It can also catalyze the
removal of 3′-phosphoryl groups from 3′-phosphoryl polynucleotides. Applications
of T4 polynucleotide kinase include labeling DNA or RNA ends with radiolabeled
ATP as a phosphate donor for probe preparation and base-specific sequencing, mapping of restriction sites, removal of 3′-phosphoryl groups, and synthesis of substrates for DNA or RNA ligase by the addition of 5′-phosphates (Wang et al. 2002).
4 Enzymes as Molecular Tools
zinc and magnesium ions and is inactivated by chelating agents (Mössner et al.
1980).
4.10.1.3 Shrimp Alkaline Phosphatase
It is a highly specific, heat-sensitive phosphatase enzyme isolated from arctic shrimp
(Pandalus borealis). Its catalytic action is similar to BAP and CIP, but being heat
sensitive, it can be irreversibly inactivated by heat treatment at 65 °C for 15mins. It
is used for 5′ dephosphorylation of DNA or RNA for subsequent labeling of the
ends, to prevent self-ligation, and for the inactivation of unused dNTPs from PCR
product prior to sequencing.
4.10.2 Acid Phosphatases
Acid phosphatases have optimal pH below 7.0. It is commercially produced from
wheat germ. Three isozymes of acid phosphatase have been isolated, namely EI,
EII, and EIII with similar molecular weights (55 kDa ± 5 kDa) and having optimum
pH’s 5.5, 4.5, and 4.0, respectively (Verjee 1969).
4.10.2.1 Tobacco Acid Pyrophosphatase
Tobacco acid pyrophosphatase (TAP) catalyzes the hydrolysis of various pyrophosphate bonds, including the triphosphate bridge of 5′-terminal methylated guanine
nucleotide cap of mRNA, generating a 5′-phosphate terminus which can be dephosphorylated with alkaline phosphatase, and labeled with T4 polynucleotide kinase
for radiolabeled probe production or ligated with oligonucleotide adaptors for
RACE. EpiCentre, the only manufacturer of TAP, has discontinued its production.
TAP was produced directly from tobacco callus tissue due to difficulties in making
a recombinant enzyme. Decapping Pyrophosphohydrolase from tebu-bio and CapClip™ Acid Pyrophosphatase from CellScript are the available replacements for
TAP of which the latter was found to have better catalytic activity.
4.10.3 T4 Polynucleotide Kinase
T4 polynucleotide kinase, isolated from T4 bacteriophage infected E. coli, is the
most widely used polynucleotide kinase in molecular biology. It is a homotetramer
of 33-kDa monomers encoded by pse T gene. T4 polynucleotide kinase has phosphatase activity at 3′ end and kinase activity at 5′ end. It catalyzes the transfer of a
phosphate group from ATP molecule to the 5′ end of double-stranded and singlestranded DNA or RNA or nucleoside 3′-monophosphates. It can also catalyze the
removal of 3′-phosphoryl groups from 3′-phosphoryl polynucleotides. Applications
of T4 polynucleotide kinase include labeling DNA or RNA ends with radiolabeled
ATP as a phosphate donor for probe preparation and base-specific sequencing, mapping of restriction sites, removal of 3′-phosphoryl groups, and synthesis of substrates for DNA or RNA ligase by the addition of 5′-phosphates (Wang et al. 2002).
4 Enzymes as Molecular Tools