PCR reaction components
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| Template |
The quality of the template influences the outcome of the PCR. For instance,
large amounts of RNA in a DNA template can chelate Mg2+ and reduce the yield
of the PCR. Also, impure templates may contain polymerase inhibitors that
decrease the efficiency of the reaction. Note: To get the purest template,
always use a purification product specifically designed to purify DNA such
as the High Pure
PCR Template Preparation Kit from Roche Applied Science (Cat. No.
1796828). The integrity of the template is also important. Template DNA
should be of high molecular weight. To check the size and quality of the
DNA, run an aliquot on an agarose gel. When testing a new template, always
include a positive control with primers that amplify a product of known
size and produce a good yield.
Example: For testing human genomic DNA, the Human
tPA Control Primer Set from Roche Applied Science (Cat. No. 1691104,
for amplification of 4.8 kb, 9.3 kb, and 15.0 kb targets) can serve as a
positive control. The amount of template in a reaction strongly influences
performance in PCR. The recommended amount of template for standard PCR
is:
- a maximum of 500ng of human genomic DNA
- 1–10 ng bacterial DNA
- 0.1–1 ng plasmid DNA
Low amounts of template, e.g. <10 ng human genomic DNA, will require
specific reaction modifications, such as changes in cycle number, redesign
of primers, use of “Hot Start”, etc. |
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| Primers |
In most PCR applications, it is the sequence and the concentration of
the primers that determine the overall assay success. For convenience, several
primer design software programs are available. These can be used to ensure
that the primer sequences have the following general characteristics:
- Are 18–24 bases long.
- Contain no internal secondary structure.
- Contain 40–60% G/C.
- Have a balanced distribution of G/C and A/T rich domains.
- Are not complementary to each other at the 3' ends (so primer-dimers
will not form).
- Have a melting temperature (Tm) that allows annealing temperatures
of 55°–65°C (for maximum specificity use temperatures of 62°–65°C).
Note: Optimal annealing temperatures are often higher than the Tm of the
primers (approx. 5°–10°C) and have to be determined empirically. Caution:
For both primers the Tm should be similar. Bases that do not hybridize to
the template may be added at the 5' end of a primer, (e.g. for introducing
restriction sites into the amplification product). Primer concentrations
between 0.1 and 0.6 µM are generally optimal. Higher primer concentrations
may promote mispriming and accumulation of nonspecific product. Lower primer
concentrations may be exhausted before the reaction is completed, resulting
in lower yields of desired product. Note: For some systems, a higher primer
concentration (up to 1 µM) may improve results. When testing new primers,
always include a positive control reaction with a template that has been
tested for function in PCR. This control shows whether the primers are working.
The Human Genomic
DNA from Roche Applied Science (Cat. No. 1691112) is a good control
template for evaluation of human primer sequences. |
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| Choice of DNA polymerase |
The choice of a DNA polymerase can profoundly affect the outcome of PCR.
For most routine PCR, Taq DNA Polymerase has long been the standard PCR
enzyme. However, Taq DNA Polymerase has its limitations. Our Product
Selection Guide will provide more information on choosing the appropriate
DNA polymerase. Here are some general guidelines:
- To increase the fidelity of PCR, use Pwo
DNA Polymerase or enzyme blends (e.g. the Expand
PCR Systems which contain blends of Taq DNA polymerase and a
thermostable polymerase with proofreading activity).
- To improve PCR yield, use the Expand
High Fidelity PCR System.
- To amplify GC-rich targets, preferably use the GC-RICH
PCR System, or the Expand
Long Template PCR System with supplied buffer 3.
- To amplify targets up to 5 kb, use the Expand
High Fidelity PCR System, or the High
Fidelity PCR Master.
- To amplify targets >5 kb, use Expand
Long Template PCR System or Expand
20 kb PLUS PCR System.
For most assays, the optimum amount of thermostable DNA polymerase (or a
blend of polymerases) will be between 0.5 and 2.5 units/50 µl reaction volume.
Increased enzyme concentrations sometimes lead to decreased specificity.
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| MgCl2 concentration |
| Mg2+ forms soluble complexes with dNTPs to produce the actual substrate
that the polymerase recognizes. The concentration of free Mg2+ depends on
the concentrations of compounds that bind the ion, including dNTP, free
pyrophosphate (PPi) and EDTA. For best results, always determine the optimal
Mg2+ concentration empirically. The optimal Mg2+ concentration may vary
from approximately 1 mM to 5 mM. The most commonly used Mg2+ concentration
is 1.5 mM (with dNTPs at a concentration of 200 µM each). Mg2+ influences
enzyme activity and increases the Tm of double-stranded DNA. Excess Mg2+
in the reaction can increase non-specific primer binding and increase the
non-specific background of the reaction. Note: For easy optimization of
MgCl2, see the
PCR Optimization Kit from Roche Applied Science (Cat. No. 1636138).
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| Deoxynucleoside triphosphate (dNTP) concentration |
Always use balanced solutions of all four dNTPs to minimize polymerase
error rate. Imbalanced dNTP mixtures will reduce Taq DNA Polymerase fidelity.
Note: For maximum convenience, a premixed, balanced mixture of dNTPs such
as the PCR
Nucleotide Mix, Cat. No. 1581295) may be added to the reaction mixture
as a single reagent. In addition, individual PCR
Grade dNTPs and a set of PCR
Grade dNTPs (Cat. No. 1969064) are available. If you increase the
concentration of dNTPs you must also increase Mg2+ concentration. Increases
in dNTP concentration reduce free Mg2+, thus interfering with polymerase
activity and decreasing primer annealing. For prevention of carry-over contamination
a higher concentration of dUTP is usually used in place of dTTP (for details,
see Preventing
carry-over contamination with uracil-DNA glycosylase). The final
dNTP concentration should be 50–500 µM (each dNTP). The most commonly used
dNTP concentration is 200 µM. |
| pH |
| Generally, the pH of the reaction buffer supplied with the corresponding
thermostable DNA polymerase (pH 8.3–9.0) will give optimal results. However,
for some systems, raising the pH may stabilize the template and enhance
results. Note: Determining the optimal reaction pH can be simplified with
the PCR Optimization
Kit from Roche Applied Science (Cat. No. 1636138). |
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| Reaction additives |
In some cases, adding the following compounds can enhance the efficiency
or specificity of PCR:
- Betaine (0.5–2 M)
- Bovine serum albumin (BSA; 100 ng/50 µl)
- Detergents
- Dimethylsulfoxide (DMSO; 2–10%) (v/v)
- Gelatine
- Glycerol (1–5%) (v/v)
- Pyrophosphatase (0.001–0.1 units/reaction)
- Spermidine
- T4 Gene 32 protein
The effect of these additives must be determined empirically, e.g. with
the PCR Optimization
Kit from Roche Applied Science (Cat. No. 1636138). |
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| Reaction overlay |
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A layer of mineral oil is often placed atop the reaction mixture to prevent
evaporation during PCR. Note: If the thermal cycler has a top heater,
oil overlay is not necessary.
Note: If the thermal cycler has a top heater, oil overlay is not necessary.
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