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Successful PCR Conditions

Parameters for Successful PCR

Are you having problems of achieving a successful PCR?  Many factors can affect your outcome of your PCR such as: Metal Ion Cofactors, Substrate and Substrate Analogs, Buffers and Salts and Cosolvents.  Also Thermal Cycling Considerations:  PCR Vessels, Temperature and Time Optimization, PCR Amplification Cycles, Enzyme/Target and Hot Start. 

Metal Ion Cofactors and PCR

An essential cofactor for the DNA polymerase in PCR is Magnesium chloride.  Its concentration must be optimized for every primer:template system. Many components of the reaction bind magnesium ion, including primers, template, PCR products and dNTPs. The main 1:1 binding agent for magnesium ion is the high concentration of dNTPs in the reaction. Because it is necessary for free magnesium ion to serve as an enzyme cofactor in PCR, the total magnesium ion concentration must exceed the total dNTP concentration. Typically, to start the optimization process, 1.5 mM magnesium chloride is added to PCR in the presence of 0.8 mM total dNTPs. This leaves about 0.7 mM free magnesium for the DNA polymerase. In general, magnesium ion should be varied in a concentration series from 1.5–4.0 mM in 0.5 mM steps.

Substrates and Substrate Analogs for PCR

The DNA polymerases incorporate very efficiently dNTPs.  They also can incorporate modified substrates, when they are used as supplemental components in PCR. Examples of substrates used for DNA polymerase are: Digoxigenin-dUTP, biotin-11-dUTP, dUTP, c7deaza-dGTP, and fluorescently labeled dNTPs. For conventional PCR, the concentration of dNTPs remains balanced in equimolar ratios, e.g., 200 μM each dNTP. Also note that, deviations from these standard recommendations may be beneficial in certain amplications. For example, when random mutagenesis of a specific target is desired, unbalanced dNTP concentrations promote a higher degree of misincorporations by the DNA polymerase.

Buffers and Salts for PCR

Depending on the DNA polymerase used the optimal PCR buffer concentration, salt concentration, and pH should be picked accordingly to the DNA polymerase. The PCR buffer for Taq DNA polymerase consists of 50 mM KCl and 10 mM Tris-HCl, pH 8.3, at room temperature. This buffer provides the ionic strength and buffering capacity needed during the reaction. It is important to note that the salt concentration affects the Tm of the primer:template duplex, and hence the annealing temperature.

Cosolvents

Different PCR Cosolvents are used to increase the yield, efficacy, and specificity of PCR amplifications. These cosolvents can be advantageous in some amplifications, and disadvantageous in other amplifications. It is impossible to predict which additive will be useful for each primer:template duplex and therefore the cosolvent must be empirically tested for each combination.

Thermal Cycling Considerations

PCR Vessels

PCR must be performed in vessels that are compatible with low amounts of enzyme and nucleic acids and that have good thermal transfer characteristics. Usually, polypropylene is used for PCR vessels and conventional, thick-walled microcentrifuge tubes are chosen for many thermal cycler systems. PCR is most often performed at a 10–100 μL reaction scale and requires the prevention of the evaporation/condensation processes in the closed reaction tube during thermal cycling. A mineral oil overlay or wax layer serves this purpose. More recently, 0.2-mL thin-walled vessels have been optimized for the PCR process and oil-free thermal cyclers have been designed that use a heated cover over the tubes held within the sample block.

Temperature and Cycle Time Optimization

It is essential that the reaction mixtures reach the denaturation, annealing, and extension temperatures in each thermal cycle. If insufficient hold time is specified at any temperature, the temperature of the sample will not be equilibrated with that of the sample block. Some thermal cycler designs time the hold interval based on the block temperature, whereas others base the hold time on predicted sample temperature. If a conventional thick-walled tube used in a cycler controlled by block temperature, a 60-s hold time is sufficient for equilibration. Extra time may be recommended at the (72°C) extension step for longer PCR products. Using a thin-walled 0.2-mL tube in a cycler controlled by predicted sample temperature, only 15 s is required. To use existing protocols or to development protocols for use at multiple laboratories, it is very important to choose hold times according to the cycler design and tube wall thickness.

PCR Amplification Cycle Number

The number of PCR amplification cycles should be optimized with respect to the starting concentration of the target DNA. It is recommended that, from 40– 45 cycles to amplify 50 target molecules, and 25–30 cycles to amplify 3 × 105 molecules to the same concentration. This non-proportionality is caused by a so-called plateau effect, in which a decrease in the exponential rate of product accumulation occurs in late stages of a PCR. This may be caused by degradation of reactants (dNTPs, enzyme); reactant depletion (primers, dNTPs); end-product inhibition (pyrophosphate formation); competition for reactants by non-specific products; or competition for primer binding by reannealing of concentrated (10 nM) product. It is usually advisable to run the minimum number of cycles needed to see the desired specific product, because unwanted nonspecific products will interfere if the number of cycles is excessive.

 Enzyme / Target

In a standard aliquot of Taq DNA polymerase used for a 100-μL reaction, there are about 1010 molecules. Each PCR sample should be evaluated for the number of target copies it contains or may contain. For example, 1 ng of lambda DNA contains 1.8 × 107 copies. For low-input copy number PCR, the enzyme becomes limiting and it may be necessary to give the extension process incrementally more time. Thermal cyclers can reliably perform this automatic segment extension procedure in order to maximize PCR yield.

Hot Start Conditions

All of the above optimizations also apply to a PCR that is designed, from the beginning, with a hot start method. Often, a hot start can be incorporated successfully into a previously optimized PCR without changing the reaction conditions. However, it usually pays to reoptimize after adding a hot start. Optimization is often a balance between producing as much product as possible and overproducing nonspecific, background amplifications. Because hot start greatly reduces background amplifications, the upper restraints are raised on conditions such as enzyme concentration, cycle number, and metal ion cofactor concentration. Sensitive PCRs that have been highly tuned without a hot start may fail when a hot start is added. This can be caused by slight delays in early cycles caused by mixing or enzyme activation. The PCR usually can be restored, often with substantial increase in specific product, by simply increasing limiting parameters or reagents. In addition, there are optimizations specific to each hot start method. Mixing or enzyme activation can be affected by PCR volume, buffer composition and pH, cosolvents, cycling conditions, and so on. The specific product’s literature, often a product insert, should be consulted for information on these considerations.

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