214
FRIEDHELM HILDEBRANDT AND IVA SINGH-SAWHNEY
from the first PCR can be destroyed with the help of uracil N -glycosylase
before the second PCR is performed (3).
Comments
Optimizing reaction conditions
• Specificity
Specificity ofPCR is on the order of 10 7 , since a specific fragment of 300bp
can be isolated from a complex template such as human genomic DNA
(3 x 10 9 bp ). Specificity ( or "stringency") is increased at high er annealing
temperatures and lower MgCh concentrations.
All PCR products, specific or non-specific, will accumulate exponentially
once the second primer anneals to an extension product of the first primer. Therefore, the first few cycles are the most critical, when trying to
avoid the generation of non -specific products. It may be advisable to
start with an initial denaturation of 4 min at 95°C to assure complete
strand separation before the first cycle, thus avoiding artifacts due to
incomplete denaturation, secondary structure formation of the template, and formation of so-called "shuffle clones" which are derived
from in vitro recombination or template strand switching.
The PCR can be performed on crude preparations ofblood or directly on
unpurified cells. In these instances Taqpolymerase can be added as the last
PCRcomponentafterinitialincubationat95°Chasbeenstarted,todenature
anyproteases thatmaybe present in the template. This "hot start" approach
will also reduce the generation of non-specific products, by avoiding unwanted primer annealing and extension at low temperatures (which may
occur when heating up the reactions from ice to 95°C). Rather than using
mineral oil, the PCR can be overlaid with a wax ( Stratagene) on top ofwhich
the Taqpolymerase is pipetted. When a temperature above 90°C is reached,
the polymerase will melt through the wax and gain contact with the reaction mixture ("hot start''). E. coli ssDNA binding protein and anti-Tagantibodies have also been used to increase specific amplification ( 4).
If too many cycles are performed, the exponential accumulation of the
specific product will plateau and non-specific products may "catch
up", thus impairing specificity as well as yield.
• Yield
Foramplificationfrom3 X 1 0 5 templatesandfrom 1 0 3 templates,asufficient
cycle n umher will be 25-30 and 40 cycles, respectively. Whereas dNTPs and
FRIEDHELM HILDEBRANDT AND IVA SINGH-SAWHNEY
from the first PCR can be destroyed with the help of uracil N -glycosylase
before the second PCR is performed (3).
Comments
Optimizing reaction conditions
• Specificity
Specificity ofPCR is on the order of 10 7 , since a specific fragment of 300bp
can be isolated from a complex template such as human genomic DNA
(3 x 10 9 bp ). Specificity ( or "stringency") is increased at high er annealing
temperatures and lower MgCh concentrations.
All PCR products, specific or non-specific, will accumulate exponentially
once the second primer anneals to an extension product of the first primer. Therefore, the first few cycles are the most critical, when trying to
avoid the generation of non -specific products. It may be advisable to
start with an initial denaturation of 4 min at 95°C to assure complete
strand separation before the first cycle, thus avoiding artifacts due to
incomplete denaturation, secondary structure formation of the template, and formation of so-called "shuffle clones" which are derived
from in vitro recombination or template strand switching.
The PCR can be performed on crude preparations ofblood or directly on
unpurified cells. In these instances Taqpolymerase can be added as the last
PCRcomponentafterinitialincubationat95°Chasbeenstarted,todenature
anyproteases thatmaybe present in the template. This "hot start" approach
will also reduce the generation of non-specific products, by avoiding unwanted primer annealing and extension at low temperatures (which may
occur when heating up the reactions from ice to 95°C). Rather than using
mineral oil, the PCR can be overlaid with a wax ( Stratagene) on top ofwhich
the Taqpolymerase is pipetted. When a temperature above 90°C is reached,
the polymerase will melt through the wax and gain contact with the reaction mixture ("hot start''). E. coli ssDNA binding protein and anti-Tagantibodies have also been used to increase specific amplification ( 4).
If too many cycles are performed, the exponential accumulation of the
specific product will plateau and non-specific products may "catch
up", thus impairing specificity as well as yield.
• Yield
Foramplificationfrom3 X 1 0 5 templatesandfrom 1 0 3 templates,asufficient
cycle n umher will be 25-30 and 40 cycles, respectively. Whereas dNTPs and