Some manufacturers have recently introduced instruments and consumables that are targeted to those performing “fast PCR”—a PCR protocol completed in less than half of the typical 90 minutes. Many researchers assume that fast PCR is obtainable only by purchasing these specialized, faster ramping thermal cyclers; however, simply modifying thermal cycling conditions produces most of the time savings.

With conventional instruments, reagents, and reaction vessels, it is possible to:
• Shorten run times for standard PCR from the usual 90 minutes to approximately 30 minutes
• Reliably amplify long targets (1 to 20 kb) 3x to 4x faster than with standard protocols
• Obtain real-time quantitative PCR (qPCR) data with SYBR Green, EvaGreen, or TaqMan chemistries in less than an hour

Saving time step by step

Standard PCR protocols for amplifying targets of <1,000 bp comprise several steps, each of which can be modified to shorten overall run times. Overall reaction time for conventional PCR can be reduced from about 90 minutes to <30 minutes by shortening hold times and by minimizing the temperature excursion between one step and the next (Figure 1). Some simple ways to shorten run times include the following:

Protocol
• Begin with this fast PCR protocol template: 98 C, 30 sec; then 35 cycles of 92 C, 1 sec, and 70 C, 15 sec; then 72 C, 1 min.
• Modify the 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 the rapid optimization strategy that uses temperature gradients to optimize both speed and specificity.
• If the starting target number is <100 copies, perform 40 cycles.

Reaction mix
• For targets <1 kb, use a hot-start polymerase; for targets >1 kb, use a high-fidelity DNA polymerase.
• If using existing primers, verify that Tm values are in the range of 58 to 72 C. If designing new primers, specify Tm values near 70 C Target DNA.

Any size PCR product up to 20 kb can be amplified using these fast PCR guidelines. The fastest reaction times can be achieved with shorter targets.

Figure 1. Reactions run in <35 min generate results comparable to those run in 90 min. S, standard protocol: 95 C for 3 min, the 35 cycles of 95 C for 15 sec, 60 C for 30 sec, and 72 C for 30 sec, followed by 72 C for 10 min. Actual run time, 88 min. F, fast protocol: 98 C for 30 sec, the 35 cycles of 92 C for 1 sec and 70 C for 15 sec, followed by 72 C for 1 min. Actual run time, 32 min. All amplicons were designed with primer Tm=68-72 C. Each 20-µL reaction contained 2,000 human genome targets.

Initial denaturation

The first step in PCR is generally performed at 94 to 96 C for 2 to 20 minutes. This step denatures the initial template into single-stranded DNA and activates hot-start polymerases. While 2 to 3 minutes at 94 to 95 C is usually sufficient to fully denature total genomic DNA, some hot-start polymerases require 15 or 20 minutes at 95 C to be activated. When using an antibody-modified hot-start polymerase, however, both activation and initial denaturation can be accomplished in just 15 to 30 seconds at 98 C (Figure 2). These parameters can also work well for qPCR with no deleterious effects on reaction efficiencies or Ct values over a range of target concentrations.

Click here to enlarge. Figure 2. Initial denaturation and enzyme activation time requires 30 sec or less with iQ supermix, which uses iTaq hot-start polymerase. Gel image shows a 505-bp ß-globin target amplified using iTaq polymerase with a range of initial denaturation conditions. Protocol included initial denaturation conditions as shown and then 35 cycles of 92 C for 1 sec and 68 C for 15 sec, followed by 72 C for 1 min. Actual run time: 34–38 min.

Because most polymerases are highly active in the temperature range typical for primer annealing (55 to 70 C), the annealing and extension steps of a PCR protocol can often be consolidated into a single step. Using a two-step PCR protocol rather than the standard three-step protocol reduce run time significantly.

Further reductions can be achieved by shortening the incubation time of this combined annealing/extension step. The standard annealing times (15 to 60 seconds) and extension times (1 minute per kb of PCR product) are, in most instances, unnecessarily long. Because primer concentrations are high, relative to template, primer annealing requires just a few seconds at the optimal reaction temperature.

Furthermore, a well-optimized reaction using a hot-start polymerase can amplify PCR products efficiently with much shorter extension times. As shown in Figure 1, 15-seconds combined annealing/extension incubation can be sufficient for PCR products up to 500 bp. Even shorter extension times are possible with a high-fidelity DNA polymerase, which can amplify a 2-kb target with an annealing/extension time of <15 seconds.

Optimizing the annealing/extension temperature is important because it is the major determinant of specificity of the reaction. If the annealing temperature is too high, the primers will not anneal efficiently, resulting in no amplification or poor yield. If it is too low, primer mismatches and nonspecific amplification may occur and yield may be diminished (Rychlik et al. 1990). To maximize both speed and specificity, the highest possible annealing temperature should be used without sacrificing adequate reaction yield. Gradient-enabled thermal cyclers allow optimization of the annealing temperature in a single run.

Denaturation while cycling

The hold times and temperatures required to denature the template during PCR cycling are not as stringent as in the initial denaturation step because the template being denatured is a PCR product, which is usually much shorter and less complex than the initial template DNA.

We have found that a 1-second denaturation at 92 C is sufficient for a variety of PCR products amplified with iQ supermix, including the 83.5% GC, 505-bp PCR product in Figures 1 and 2, as well as a 64% GC, 150-bp PCR product in lambda DNA. This is consistent with the observation of Yap and McGee (1991) that temperatures above 92 C are unnecessary for denaturing PCR products shorter than 500 bp.

Table. Real-time qPCR with dual-labeled probes completed in less than an hour. Results are shown for real-time PCR assays using dual-labeled probes. Identical reactions amplifying ß-actin from human liver cDNA using iTaq supermix with ROX were run on the iQ5 real-time system using different thermal cycling conditions.

To establish general considerations (see Table) for choosing primers and annealing/extension temperatures for fast PCR, we performed a series of reactions using a range of annealing/extension temperatures and a panel of primer pairs that had average Tm values varying from 58 to 72 C. The fastest overall reaction times for each primer pair were obtained by using the highest annealing/extension temperature that generated a good band on the gel (i.e. strong intensity, single product).

Ramping time

Ramping time is the time required by the thermal cycler to transition from one incubation temperature to another. Two parameters contribute to ramping time—the ramp rate of the cycler and the difference between consecutive temperatures. Smaller temperature excursions require shorter ramping times.

While the contribution of ramp rate to overall cycling time has been highlighted by manufacturers of faster-ramping thermal cyclers, the time saved by using these specialized instruments is relatively minor (6 to 8 minutes) compared to the savings gained from optimizing thermal cycling parameters for speed (56 to 65 minutes).

Ramping time can also be reduced by converting from a three-step to a two-step protocol in which the annealing and extension steps are combined at a temperature optimal for primer annealing yet sufficient for primer extension. Such two-step PCR protocols generate yields similar to three-step protocols for products up to 200 bp (Cha and Thilly 1995). Furthermore, a combined annealing and extension step at 60 C is typical for qPCR assays using TaqMan probes, and reaction efficiencies of about 100% are routinely achieved for such assays. This suggests that the processivity of <i>Taq</i> at this lower temperature is sufficient to extend fully products of 70 to 200 bp.

Click here to enlarge. Figure 3. The final extension step can be reduced to 1 min or less. Targets of 164–1,037 bp were amplified from human genomic DNA using a fast PCR protocol. Then a final step of 0 to 5 min at 72 C was performed before gel analysis. Cycling protocol for 164-bp PCR product: 98 C, 30 sec; then 35 cycles of 92 C, 1 sec and 68 C, 15 sec. Actual run time, 33–38 min. Cycling protocol for 505 and 1,037-bp PCR products: 98 C, 30 sec; then 35 cycles of 92 C, 1 sec and 68 C, 30 sec. Actual run time: 41–46 min.

A post-PCR final incubation step of 5 to 10 minutes at 72 C is often recommended to promote complete synthesis of all PCR products. Although this is commonly referred to as an extension step, a major purpose is to allow reannealing of the PCR product into double-stranded DNA, so it can be used for cloning or visualized using ethidium bromide after gel electrophoresis. This step can be shortened to 30 to 60 seconds for PCR products of 100 to 1,000 bp (Figure 3).

Saving time in long PCR

In general, longer targets (>1 kb) need longer extension times, resulting in runs that can last several hours. High-performance enzymes have been developed that exhibit improved speed and robustness and provide increased product yield. Through the use of patented (#6,627,42) Sso7d fusion protein technology, these enzymes are able to amplify very long DNA fragments (up to 20 kb) while providing significant time savings up to 3- and 4-fold.

Viresh Patel, viresh_patel@bio-rad.com, directs product development in the areas of reverse transcription and qPCR and began his career in the Life Science industry at Ingenuity Systems after completing his Ph.D. from the University of California, Los Angeles, in 2000.

References

• Breslauer, K. J., et al. 1986. Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci USA. 83: 3746–3750
• Cha, R. S., and W. G Thilly. 1995. Specificity, efficiency, and fidelity of PCR. In PCR Primer: A Laboratory Manual, ed. C.W. Dieffenbach and G.S. Dveksler, 37–52. New York: Cold Spring Harbor Laboratory Press.
• Rychlik, W., et al. 1990. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 18: 6409–6412.
• Rozen, S., and H. J. Skaletsky. 2000. Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics Methods and Protocols: Methods in Molecular Biology, ed. S. Krawetz and S. Misener, 365–386. New Jersey: Humana Press. Source code available at http://fokker.wi.mit.edu/primer3/
• Yap, E. P., and J. O. McGee. 1991. Short PCR product yields improved by lower denaturation temperatures. Nucleic Acids Res. 19: 1713.