14 Polymerase Chain Reaction
215
primers are used in large excess, yield is mostly determined by amount of
template and activity of Taq polymerase (see below).
• Polymerase processivity
The processivity of the polymerase is defined as the rate of nucleotide
incorporation at 72°C is approximately 35 to 100 nt/sec (5). Whereas
the record for PCR product length currently lies araund 30 kb, fragments
ofup to 3 kb be amplifiedroutinely. Processivitycan beimproved byadding
accessoryproteins (such as bovine serum albumin at 100 ~glml), lowering
the salt concentration, or by using novel DNA polymerases.
• Polymerase fidelity
The misincorporation rate for Taq polymerase is thought tobe on the order
of 10- 4 -10- 5 /cycle. Misincorporation is reduced by decreasing MgCh
concentration, decreasing dNTP concentration, raising annealing
temperatures, and decreasing extension times. Fidelity will, however,
be impaired at dNTP concentrations <10 ~M or if concentrations of
the four dNTPs differ. The authentic sequence of a target region can
be determined despite some misincorporation by sequencing several
independent PCR products and generating a consensus sequence.
• Taq polymerase
The concentration of Taq polymerase can be optimized for a template
using 0.5 to 5 U/100~1 and monitaring the PCR product yield by agarase
gel electrophoresis. If insufficient enzyme is used, low yield will result.
Excessive enzymewill give rise to non-specific PCR products. The activity
of the enzyme varies by 2 orders of magnitude between 20 and 85°C. The
half-life ofTaq polymerase activity is estimated tobe >2 h, 40 min, and
5 min at 92.5°C, 95°C, and 97.5°C, respectively.
• dNTPs
dNTPstocks(10mM)arestoredat-20°C.ConcentrationofeachdNTPcanbe
altered between 10 ~M and 50 ~M (up to 200 ~M) for optimal results.
Decreasing the amount of dNTPs should result in improved specificity
and fidelity (6). However, fidelity will be less at dNTP concentrations
<10 ~M or if concentrations of the four dNTPs differ.
• Magnesium
Magnesium concentration influences product specificity, enzyme processivity and fidelty, and specificity of primer annealing. The optimal magnesiumconcentrationmayvarywithdifferenttemplatesandprimerpairs.It
can be altered from 0.5 to 10 mM, the standard being 1.5 mM.
215
primers are used in large excess, yield is mostly determined by amount of
template and activity of Taq polymerase (see below).
• Polymerase processivity
The processivity of the polymerase is defined as the rate of nucleotide
incorporation at 72°C is approximately 35 to 100 nt/sec (5). Whereas
the record for PCR product length currently lies araund 30 kb, fragments
ofup to 3 kb be amplifiedroutinely. Processivitycan beimproved byadding
accessoryproteins (such as bovine serum albumin at 100 ~glml), lowering
the salt concentration, or by using novel DNA polymerases.
• Polymerase fidelity
The misincorporation rate for Taq polymerase is thought tobe on the order
of 10- 4 -10- 5 /cycle. Misincorporation is reduced by decreasing MgCh
concentration, decreasing dNTP concentration, raising annealing
temperatures, and decreasing extension times. Fidelity will, however,
be impaired at dNTP concentrations <10 ~M or if concentrations of
the four dNTPs differ. The authentic sequence of a target region can
be determined despite some misincorporation by sequencing several
independent PCR products and generating a consensus sequence.
• Taq polymerase
The concentration of Taq polymerase can be optimized for a template
using 0.5 to 5 U/100~1 and monitaring the PCR product yield by agarase
gel electrophoresis. If insufficient enzyme is used, low yield will result.
Excessive enzymewill give rise to non-specific PCR products. The activity
of the enzyme varies by 2 orders of magnitude between 20 and 85°C. The
half-life ofTaq polymerase activity is estimated tobe >2 h, 40 min, and
5 min at 92.5°C, 95°C, and 97.5°C, respectively.
• dNTPs
dNTPstocks(10mM)arestoredat-20°C.ConcentrationofeachdNTPcanbe
altered between 10 ~M and 50 ~M (up to 200 ~M) for optimal results.
Decreasing the amount of dNTPs should result in improved specificity
and fidelity (6). However, fidelity will be less at dNTP concentrations
<10 ~M or if concentrations of the four dNTPs differ.
• Magnesium
Magnesium concentration influences product specificity, enzyme processivity and fidelty, and specificity of primer annealing. The optimal magnesiumconcentrationmayvarywithdifferenttemplatesandprimerpairs.It
can be altered from 0.5 to 10 mM, the standard being 1.5 mM.