Converting an existing PCR assay to a Fast PCR assay that requires significantly less thermocycler time is an attractive proposition, but may end up costing time and reagents if the conversion compromises reaction performance (sensitivity, yield or reproducible amplification). Before using this (or any other) Fast PCR kit, it is therefore important to understand the basic principles of Fast PCR.
A PCR cycle is based on three processes: denaturation, primer annealing and extension. The relative contribution of each of these to the total reaction time depends on a number of factors, and suboptimal execution of any step may decrease the efficiency of the reaction, leading to lower yield of the target amplicon, reduced sensitivity and/or inconsistent amplification.
After initial denaturation of the template DNA, complete denaturation of product formed in each cycle is necessary for the exponential accumulation of the target amplicon. Denaturation is typically conducted at 94-96°C and the efficiency of this step is determined by efficiency of heat transfer to the reaction components and the nature of the DNA template and fragment that is being amplified. Thin-walled PCR tubes and plates facilitate heat transfer, whereas large reaction volumes reduce its efficiency. Complex templates and amplicons with a high GC-content and/or stable secondary structure usually require longer denaturation times and/or higher temperatures to ensure that the maximum number of template molecules are available during each successive cycle.
Exponential accumulation of new amplicon molecules depends on the efficient annealing of primers to each available template molecule during each successive PCR cycle. Complex templates (e.g. genomic DNA) require longer annealing times than simple ones (e.g. plasmid, phage or bacmid DNA), and certain primer types (e.g. those with a low GC-content) may require longer annealing times. The effective annealing time is determined by the characteristics of the particular primer pair. Primers with a broad annealing temperature range result in longer effective annealing times than primers with sharply defined optimal annealing temperatures (see Sections 4.2 and 4.3 for more details).
Exponential accumulation of the desired amplicon finally depends on full extension of each annealed primer molecule by the DNA polymerase. Conventional protocols with Taq DNA polymerase provide for 1 min/kb extension time at the optimal temperature for Taq polymerase activity (72°C). The relative contribution of extension time to the total reaction time increases with increasing amplicon size (Table 1).
Table 1: Total cycle extension time as a percentage of total reaction time for different amplicon lengths in a conventional PCR assay with wild-type Taq
Figures are based on a 35-cycle reaction profile consisting of an initial denaturing step of 2 min at 95°C; 15 sec denaturing (95°C), 15 sec annealing (60°C) and 1 min/kb extension (72°C) per cycle, and a final extension at 72°C of 30 sec/kb. Thermocycler ramp rates of 2oC/sec heating and 1oC/sec cooling were used in calculations.
The amount of time that can be saved by using competitor Fast PCR kits and protocols based on wild-type Taq is limited by the extension rate of the enzyme. To achieve shorter assay times the denaturation and annealing times in each cycle have to be shortened. Special ultra-thin plastics and reduced reaction volumes allow for improved heat transfer during denaturation, and specially formulated buffers are employed to increase the efficiency of primer annealing, thereby reducing annealing times. In addition, extension times are shortened, particularly for short amplicons. However, such “artificially” shortened reaction profiles often result in reduced efficiency of one or more of the cycling processes, thereby reducing the yield of the target amplicon and/or sensitivity of the assay. Results comparable to those obtained with the original assay are consequently only achievable in assays with short amplicons and very high target copy numbers.
In contrast, the KAPA2G Fast PCR kit is based on an engineered polymerase with an intrinsic ability to synthesize DNA faster than wild-type Taq. The protocols outlined in Section 6 are therefore primarily based on reduced extension times that allow for a reduction of 20% to more than 70% of the original PCR time without the risk of compromising reaction performance.
Fast vs. slow thermocyclers
The time it takes to complete the same cycling profile on different thermocyclers differs significantly and this “real” or elapsed time (vs. programmed time) is determined by the thermal ramping rates of individual instruments (Table 2). The majority of conventional (Peltier-based) thermocyclers are only capable of heating the block at 1-1.5°C/sec. Cooling is always slower and rates of less than 1°C/sec are typical. Silver and gold (plated) blocks used in fast ramping instruments developed in recent years are capable of heating rates of 3-6°C/sec and maximum cooling rates of just more than 4°C/sec.
Table 2: Approximate assay times for the amplification of different sized amplicons with wild-type Taq using thermocyclers with different thermal ramping rates
Figures are based on a 35-cycle reaction profile consisting of an initial denaturing step of 2 min at 95°C; 15 sec denaturing (95°C), 15 sec annealing (60°C) and 1 min/kb extension (72°C) per cycle, and a final extension at 72°C of 30 sec/kb.
The KAPA2G Fast PCR Kit may be used in conjunction with any conventional thermocycler irrespective of ramp rates. The same assay will take longer to complete on a slow ramping than on a fast ramping instrument. The relative amount of time saved by converting your existing assay into a Fast assay using this kit is typically between 20 and >60%, for slow ramping instruments and between 35 and >70% for when fast ramping cyclers are used (see Section 6 for more details).
When converting your existing assay to a Fast assay, or designing a new Fast assay, it is important to take the thermal ramping characteristics of your specific thermocycler into account, as the “real” time spent on each process in each cycle (vs. the programmed time) is longer on slower ramping instruments.
For example, for a fast ramping instrument (+6°C/sec; -4°C/sec), the time spent to cool the instrument down between denaturing at 95°C and annealing at 60°C is 8.75 sec, whereas it takes almost 47 sec to achieve the same on a slow ramping (+1.5°C/sec; -0.75°C/sec) cycler. Part of this cooling time will contribute to template denaturation, and part to primer annealing. For heating the block between the annealing (60°C) and extension (72°C) steps, the times are approximately 3.8 and 8 sec for the same fast and slow instruments, respectively. Because of this “extra” time added to each of the cycling processes in each cycle, the programmed times for slower ramping instruments may be slightly shorter than those for fast ramping cyclers (see product Technical Data Sheet for detailed cycling profiles). For the same programmed times, the longer “real” cycle times of slower ramping instruments often translate into higher yields and/or sensitivity, particularly for long amplicons.
Primer design and assay times
Primer design is an important factor in Fast PCR. Guidelines for optimal primer design are widely available in printed and online resources and should be followed as far as possible. Different algorithms are available for calculating the melting temperature (Tm) of a primer and an annealing temperature (Ta) 3-5°C than the Tm is usually a good first approximation. Primer design programs and Tm calculators are, however, unable to predict the behavior of a particular primer pair with a particular template under specific reaction conditions, namely whether the primer pair will anneal over a broad Ta range or display a sharp optimal Ta. A Ta gradient PCR is therefore recommended as the first step in the optimization of any PCR assay, particularly those that are to be converted to Fast assays.
Primers with a sharply defined optimal Ta are used in traditional 3-step cycling profiles (where each cycle is programmed with a specific time for denaturation, annealing and extension). Primers that anneal efficiently over a broad Ta range (particularly in the range of 65-72°C) may be used in 2-step cycling profiles, where the annealing and extension step is combined into a single process and each cycle consists of only two specified times. When converting an existing PCR assay to a Fast assay, a significant amount of reaction time may be saved if the specific primer pair is suitable for a 2-step protocol. This should be taken into consideration when new assays are designed. More details about 2- and 3-step cycling profiles and the conversion of existing cycling profiles can be found in the product Technical Data Sheet.