Biocompare - Fast PCR Tutorial

Introduction
The polymerase chain reaction (PCR) has traditionally been optimized for specificity and, to a lesser extent, product yield. The speed with which the reaction is completed has been of secondary importance. This has changed due to the availability of software to aid in primer and amplicon design and the use of reagents that can tolerate a range of reaction conditions. This has allowed researchers to enhance their efficiency by minimizing PCR cycling times and thus maximizing throughput.

While the term "fast PCR" is not well defined, it generally describes the completion of a PCR protocol in far less time than the typical 90 minutes a standard PCR reaction takes. In fast PCR completion of a reaction in less than 40 minutes is possible for the majority of applications.

Many researchers suppose that fast PCR can be achieved only by using specialized thermal cyclers with fast ramping rates. In this tutorial, we demonstrate that, in fact, most of the time savings in fast PCR are made simply by modifying thermal cycling conditions. We also present guidelines for accomplishing fast PCR without a fast-ramping thermal cycler. Finally, we demonstrate that with conventional instruments, reagents, and reaction vessels it is possible to:

• Shorten run times for standard PCRs from around 1.5 hours to 35 minutes
• Reliably amplify long (1 to 20 kb) up to 3-fold faster than with standard protocols and enzymes, and
• Obtain SYBR Green or Taqman real-time quantitative PCR (qPCR) data in under 1 hour.

Modifications for PCR Run Times
Most PCR protocols for amplifying targets of less than 1000 bp are comprised of several denaturation, annealing and extension steps. In Fast PCR, each of these steps can be modified to shorten the overall reaction time. The overall goal is to reduce the hold time at each step and to reduce the temperature excursion between each step. A simple set of guidelines are provided here.

Protocol
There are a number of simple modifications to standard PCR protocols that can be made to decrease run times.

• A good template to begin with for a fast PCR protocol is: 98°C for 30 sec (92°C for 1 sec; 70°C for 15 sec) x 35 cycles, 72°C for 1 min.
• Set the primer annealing/extension temperature so that it is halfway between 72°C and the average of the primer Tm values. For example, if the average primer Tm is 58°C, use an annealing/extension temperature of 65°C.
• Alternatively, employ a rapid optimization strategy that uses a temperature gradient to optimize both speed and specificity.
• If the starting target number might be 100 copies, do 40 cycles.

• For targets < 1 kb, use antibody-mediated hot start polymerases. For targets >1 kb, use highly processive polymerases (iProof).
• If using existing primers, ensure the Tm values are in the range of 58-72°C. If designing new primers, specify Tm values near 70°C.

• Any size amplicon up to 20 kb can be amplified using these fast PCR guidelines and a suitable polymerase. For fastest reactions, amplify targets <250 bp.

Fast PCR — Denaturation and Activation
The goal in Fast PCR is to shorten the overall reaction time. This is accomplished by reducing the temperature differential between steps and by decreasing the hold times for each step during the PCR process.

In Fast PCR, the typical conditions for the initial denaturation/enzyme activation step would be 98°C for 30 seconds. This is in contrast to a standard PCR reaction where the initial denaturation/enzyme activation step would take place at 95°C for 10 minutes.

Fast PCR — Cycling Denaturation
The denaturation step after each cycle in Fast PCR is shorter than for standard PCR and the step occurs at a slightly lower temperature. A typical Fast PCR denaturation step lasts for 1 sec at 92°C. By comparison, a standard PCR reaction requires at least a 15 sec denaturation at 95°C.

Fast PCR — Cycling Annealing/Extension
In most Fast PCR protocols, the annealing and extension steps are combined. Because primer concentrations are high, relative to the concentration of template DNA, the annealing times for standard PCR are unnecessarily long. And since primers anneal to templates at the same temperature that taq polymerase is active, most Fast PCR reactions can combine the annealing and extension steps into one.

The annealing step is generally subject to the most optimization as it is the major determinant of specificity of the reaction. As a general guideline for choosing primers and annealing temperatures for fast PCR, those primers with Tm values of 70-72°C tend to work best.

As mentioned previously, in Fast PCR the extension and annealing steps are combined, which drastically cuts down the overall reaction time. In contrast, in a typical PCR reaction, the 30 sec annealing step is followed by a 30 sec extension step.

Fast PCR — Final Extension
In Fast PCR, this final incubation step can be shortened to 30-60 sec and is not required for amplicons less than 250 base pairs. A final incubation step for 5-10 minutes at 72°C is often recommended to promote complete synthesis of all PCR products.

Ideally the amount of product doubles in each cycle through the exponential phase of the PCR reaction. Therefore, relatively few cycles are required if the starting target concentration is high. Starting with 100 copies of target DNA, 35 cycles of PCR are adequate to detect the resulting product on an ethidium-bromide-stained gel.

It is possible to perform Fast PCR to reliably amplify long stretches of DNA. This can be done in 1/3 of the time required by standard PCR by cutting down ramp times, hold times and combining the annealing and extension steps.

Results — Short Target PCR
The results obtained with Fast PCR are comparable to those obtained with standard PCR. Here, PCR reactions run under either Fast or standard conditions are compared on an ethidium bromide stained agarose gel. The products range in size from 164 to 505 base pairs. As you can see, the results are similar between the two methods of amplification even though the Fast PCR was performed in just 0 hours 32 minutes while the standard PCR took 1 hour and 22 minutes (almost 3 times as long).

Results — Long Target PCR
Fast PCR also works with long targets. Using a high-fidelity polymerase, targets of 1-20 kilobases can be amplified 3-4 times faster using Fast PCR than when using standard PCR. Here, targets of differing lengths were amplified from lambda DNA using primers designed with a Tm = 70-72°C, so that a two-step protocol could be incorporated. Protocols were run using a range of annealing/extension times and results were scored as positive when a band of the appropriate size could be visualized on an agarose gel stained with ethidium bromide.

Results — Real Time PCR
In addition to amplifying both short and long targets, Fast PCR can be used for Real Time or quantitative PCR. For qPCR, primers are usually designed to amplify relatively short targets (70-200 bp) to ensure maximal efficiency. Such short targets may not require long denaturation and extension times, making them particulary suitable for modification for fast PCR assays without the need for special reagents, plastics, or instrumentation.

These results show the accuracy of using fast PCR protocols for Real Time analysis for a 150 bp lambda target amplified with SYBR Green Supermix. In this assay, primer concentrations were increased to 0.75 M. As this figure demonstrates, it is possible to obtain reproducible results, minimal variance around the standard curve, and reaction efficiencies close to 100% for real-time PCRs completed in under 40 minutes. For SYBR Green assays, we recommend running a post-amplification melt curve analysis. This will lengthen the overall run time by approximately 10 minutes, but will provide valuable data on reaction specificity.

Quantitative PCR using dual-labeled probes (often called TaqMan or 5' nuclease assays) incorporates a two-step PCR protocol with a combined annealing/extension step, commonly performed at 60°C. A combined annealing and extension step is necessary as the fluorescence chemistry requires the probe to be annealed to its target while the product is being extended. Again, significant run time reductions can be made simply by reducing hold times at each step.

This table is data from a TaqMan real-time qPCR run under three different thermal cycling conditions. All three reactions used the iTaq Supermix with ROX. The reaction conditions used for the "unmodified protocol" yielded a run time of 68 minutes. The run time was reduced by 7 minutes when running the reaction using a "Fast PCR" protocol that included a 3 second denaturation step and a 30 second combined annealing/extension step. A further reduction of 14 minutes was achieved by using a 30 second initial denaturation and enzyme activation step at 98°C, and reducing hold times while cycling to 1 second (at 92°C) for the denaturation step and 15 seconds for the combined annealing/extension step, resulting in an overall run time of 47 minutes. Each of these runs produced virtually identical results, with the maximum difference in average Ct between runs being 0.5 or less.

Conclusions
As with any important scientific technique, after inception, the natural evolution of the protocol is towards efficiency and accuracy. The development of Fast PCR now offers researchers a new protocol for DNA amplification and quantitation with significantly less time constraint and high quality results. Through protocol specific modifications in sample preparation and the use of versatile enzymes and reagents, the time requirement for PCR amplification can be reduced from hours to under 40 minutes. If time is truly a limiting factor in your research, then this new technique can help you begin to get back precious hours lost to arduous protocols.

Additional Results — Initial Denaturation/Enzyme Activation Step
Initial denaturation and enzyme activation requires 30 seconds or less.

Using an antibody-mediated hot start polymerase (iTaq), a 505 bp ß-globin target was amplified with a range of initial denaturation and activation conditions. Cycling profile after initial denaturation and activation was (92°C, 1 sec; 68°C, 15 sec) x 35 cycles; 72°C, 1 min. Actual run time, 34-38 min.

Additional Results — Annealing/Extension Step
Use of a thermal gradient to determine the optimal annealing/extension temperature for fast PCR reactions

The cycling profile used was 98°C, 30 sec; (92°C, 1 sec; annealing/extension temperature, 15 sec) x 35; 72°C, 1 min. Actual run time, 33-42 min. Arrows indicate the reaction conditions that will provide the shortest ramp times and highest specificity while maintaining good yield.

Additional Results — Final Extension Step
The final "extension" step can be reduced to 30-60 seconds.

Targets of 164-1037 bp were amplified from human genomic DNA using a fast PCR protocol, then a final "extension" step of 0-5 minutes at 72°C was performed before gel analysis. final extension times at 72°C before gel analysis. Cycling profile for 164 and 210 bp amplicons: 98°C, 30 sec; (92°C, 1 sec; 68°C, 15 sec) x 35. Actual run time, 33-38 min. Cycling profile for 506 and 1037 bp amplicons: 98°C, 30 sec; (92°C, 1 sec; 68°C, 30 sec) x 35. Actual run time, 41-46 min.

Troubleshooting Tips

We recommend designing and verifying primer Tm values with a calculator such as Primer-3 (http://frodo.wi.mit.edu/cgi-bin/primer3/ primer3_www.cgi) (Rozen and Skaletsky, 2000), which references the thermodynamic parameters of Breslaur (1986) and Rychlik (1990).

References

• BreslauerKJ et al., Predicting DNA duplex stability from the base sequence, Proc Natl Acad Sci USA 83, 3746-3750 (1986)
• Cha RS and Thilly WG, Specificity, efficiency, and fidelity of PCR, pp 37-52 in Dieffenbach CW and Dveksler GS (eds), PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1995)
• Innis MA and Gelfand DH, Optimization of PCRs, pp 3-12 in Innis MA et al. (eds) PCR Protocols: A Guide to Methods and Applications, Academic Press, New York (1990)
• Innis MA et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, New York (1990)
• Rychlik W et al., Optimization of the annealing temperature for DNA amplification in vitro, Nucleic Acids Res 18, 6409-6412 (1990)
• Rozen, S. and Skaletsky, H.J. (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386
• Source code available at http://fokker.wi.mit.edu/primer3/.
• Yap EP and McGee JO, Short PCR product yields improved by lower denaturation temperatures, Nucleic Acids Res 19, 1713 (1991)