Biology Reference
In-Depth Information
Table 8.2: Optimizing Standard PCR Protocols Involves Optimizing Reaction Components.
PCR component
Issues to consider
Primers
1.
Select primers with a random base distribution and GC content similar to
template DNA being amplified.
2.
Avoid primers with stretches of polypurines and polypyrimidines or other unusual
sequences.
3.
Check primers for complementarity and avoid primers with 3
′
overlaps to reduce
primer-dimer artifacts.
4.
Construct primers 20-30 nucleotides long.
5.
Optimize the amount of primers used.
6.
Design so the base at the 3
′
end of the primer is a G or C to enhance specificity
(G-C clamp).
Template DNA
1.
Template DNA should be free of proteases that could degrade the DNA
polymerase.
2.
Template DNA with high levels of proteins or salts should be diluted or cleaned up
to reduce inhibition of DNA polymerase activity.
3.
Highly concentrated template DNA may yield nonspecific product or inhibit the
reaction.
4.
It is rare that template DNA concentration is too low.
PCR buffer
1.
MgCl
2
concentration is very important.
2.
Excess Mg
2
+
promotes production of nonspecific product and primer-dimer
artifacts.
3.
Insufficient Mg
2
+
reduces yields.
4.
Presence of EDTA or other chelators can reduce the availability of Mg
2
+
.
Taq
polymerase
1.
Excessive
Taq
concentrations can yield nonspecific products and reduce yield.
Recommended concentrations are between 0.5 and 2.5 units per 100-ml reaction.
Add the
Taq
at 94 °C and mix thoroughly.
2.
Stringency can be increased by increasing the annealing temperature, adjusting
dNTP concentrations, and minimizing incubation time.
dNTPs
1.
dNTP concentrations should be equivalent to minimize misincorporation errors.
2.
Low dNTP concentrations minimize mispriming, but if too low, can reduce the
amount of product.
Cycle parameters
Incubation
1.
Time varies with length of target being amplified; 1 minute/kb is average.
2.
Ramp time (time to change from one temperature to another) should be
minimized to improve specificity.
3.
Insufficient step is a common problem; 94 °C results in complete separation, but
excess time can cause denaturation of
Taq
polymerase.
(
Continued
)
