By Jonathan Shum, Joyclyn Yee, Elena Hidalgo Ashrafi, and Natasha Paul, Ph.D.
The Polymerase Chain Reaction (PCR) is a commonplace molecular biological method to amplify a DNA target of interest. Although PCR is a powerful technique, its sensitivity and reproducibility are often plagued by off-target amplifications. These include primer dimer and mis-priming products caused by the hybridization of primers to one another or to non-specific regions on the template. Such off-target amplifications lower the efficiency of PCR by effectively sequestering PCR substrates, such as the primers, the DNA polymerase, and the dNTPs, from amplifying the desired template. At lower template concentrations, the problem of primer dimer formation and mis-priming is exacerbated as less template is available causing an increase in non-desired primer hybridization and extension.(1) Therefore, it is critical to substantially decrease or even eliminate off target amplification, especially when available template is limited.
Attempts have been made to alleviate the problem of primer dimer formation by the use of Hot Start technologies, which include physical separation of reaction components, inhibition of the DNA polymerase, and the use of accessory proteins. Many of the specialized DNA polymerase compositions can add significant cost to the reaction by the need for extensive manipulations, such as in the preparation of the DNA polymerase. In contrast, primers, which have CleanAmp™ thermolabile protecting groups, represent a simple approach to Hot Start activation in PCR. These modifications can be easily introduced to any primer sequence using standard solid phase oligonucleotide synthesis protocols. In addition, CleanAmp™ modifications are compatible with many commonly used
The introduction of thermolabile CleanAmp™ modifications into PCR primers allows for greater control of primer hybridization and extension during PCR. The thermolabile primer modifications prevent DNA polymerase extension at lower less-discriminating temperatures of reaction set-up and manipulation but also display the flexibility to allow intended extension after activation at higher temperatures (Figure 1).
Furthermore, by using either the slow-releasing Precision primers or the faster-releasing Turbo Primers, the rate of formation of unmodified primer can be controlled to suit your reaction needs. Herein, we investigate the utility of the CleanAmp™ Primer modifications in standard PCR protocols and in more advanced techniques such as multiplex PCR, one-step RT-PCR, and fast cycling PCR.
Primer dimer formation has been found to be problematic in the amplification of a region of the HIV-1 tat genomic DNA.(1) In these studies, the reaction progression was monitored by removing aliquots after 30, 35, and 40 thermal cycles (Figure 2). Amplifications using unmodified PCR primers were found to be prone to robust primer dimer formation, which competes with the formation of the desired 365 bp amplicon (Figure 2A). By contrast, the introduction of Turbo Primers significantly reduced primer dimer formation and promoted an even greater target yield as compared to the unmodified primers (Figure 2B). For Turbo Primers, only a slight amount of primer dimer is seen after 40 cycles. In the same system, the use of Precision Primers yielded only the desired amplicon, with no detectable primer dimer formation. However, while the slower release of the Precision protecting groups significantly reduced primer dimer formation, robust target amplification was slightly delayed at thirty thermal cycles but fully recovered after forty thermal cycles (Figure 2C). These studies demonstrated that Turbo Primers were able to efficiently form the desired amplicon, while significantly reducing primer dimer formation. Precision Primers were found to have the greatest utility when pure amplicon formation is required.
Mis-priming can also be a significant hindrance to the fidelity and efficiency of amplification of the desired target. In comparison to unmodified primers, Turbo Primers reduce much of the mis-priming products, with Precision Primers providing the greatest benefit. This reduction in off-target amplification is evident over a wide range of input template concentrations, with improved amplicon yield also being much greater than with unmodified primers.
Detection of a target at low concentrations is another difficulty encountered in PCR. Often, at low template concentrations, off-target amplifications compete with the desired amplification, complicating real-time PCR detection of the desired amplicon formation. CleanAmp™ Primers have been found to successfully amplify the correct amplicon at 10-100-fold lower template concentration as compared to unmodified primers. In Figure 3, the lower limit of detection of a 533 base pair amplicon from Lambda genomic DNA was explored over a range of input template concentrations using SYBR Green detection in real-time PCR. For the unmodified primers, the range of detection started above 500 copies. Because the amplification curve coincided with the no template control (NTC) curve at 500 copies or less, it would be impossible to differentiate between desired amplicon formation and other off target formation.
The use of Turbo Primers provides at least a ten-fold increase in detection, as the 50 copy concentration is distinguishable from the NTC curve. Precision Primers ultimately provide the greatest level of detection, detecting as low as 5 copies. This increased limit of detection using Precision Primers is indicative of their utility in a number of high-sensitivity downstream applications, such as single molecule detection.
Since CleanAmp™ Primers provide significant benefit relative to reactions with unmodified primers, experiments were then performed to compare the performance of CleanAmp™ Primers to that of other Hot Start technologies. In these studies, the performance of unmodified primers with one of a series of Hot Start DNA polymerases, such as a chemically modified version of Taq (2), was compared to the performance of CleanAmp™ Primers with unmodified Taq DNA polymerase. Amplicon was formed with equal or much lower yield than reactions that employed unmodified Taq DNA polymerase with Precision Primers (Figure 4A, B). Moreover, Turbo Primers and unmodified Taq DNA polymerase gave the greatest benefit, as the amplicon yield was much higher than each of the Hot Start polymerases examined. These findings are significant, as they indicate that CleanAmp™ Primers and unmodified Taq DNA polymerase can be employed without compromising amplicon yield, while efficiently reducing primer dimer formation and mis-priming.
Taq DNA polymerase was used as a point of reference to determine whether Precision and Turbo CleanAmp™ Primers could be employed with other DNA polymerases in endpoint PCR experiments. In the endpoint reactions, seven DNA polymerases devoid of Hot Start activation were evaluated for their ability to robustly form the desired 365 bp amplicon (Fig. 5). Each of the DNA polymerases examined was able to support efficient amplification of the DNA target. For all cases, with the exception of Deep Vent™ and Tfi polymerase, the units of DNA polymerase were kept constant. Overall, CleanAmp™ Primers are versatile as they can be used with other thermostable DNA polymerases in endpoint PCR. This compatibility gives great potential for the implementation of CleanAmp™ Primers in a number of applications.
One promising application of PCR is the ability to amplify multiple targets in a single reaction. This approach, known as multiplex PCR, employs a distinct primer pair for each amplicon of interest. This application has been an essential tool for many different medical diagnostic and scientific applications, such as viral screens(3), where PCR based assays have proven to be more sensitive and less time consuming than traditional cell culture tests(4).
Although multiplex PCR has many advantages, there are inherent problems that inhibit robust amplification. One major factor is competing, undesired off-target amplifications such as primer dimer formation (5), which limits the number of possible targets that can be detected per reaction, due to the increase in the number of unique primer sequences in the reaction, which in turn increases the probability of primer dimer formation. Another challenge of multiplex PCR is the preferential amplification of certain targets (6). Therefore the design of multiple primer pairs that are both specific for a target of interest and exhibit a low level of off-target amplicon formation can be a challenge. Coupled with this decreased flexibility in primer design, individual primer pair concentrations must be optimized, such that amplification efficiencies of all targets are similar (7). This is a time consuming process, which has a low probability of success, should off-target amplicon formation dominate the reaction. Below, the ability of CleanAmp™ Primers to improve the specificity of amplicon formation for all targets in multiplex PCR is evaluated. The ability of CleanAmp™ Primers to reduce other competing off-target amplification, in single target reactions was applied to a multiplex PCR assay, where amplicon yield and PCR efficiency are extremely sensitive to primer dimer formation. Findings revealed minimal optimization of the design and concentration of the CleanAmp™ Primers with CleanAmp™ Turbo Primers, improving with reduced primer dimer formation.
When individual assays are combined into a single, multiplexed PCR assay, often template concentration must be increased to compensate for inefficient amplification. However, in clinical settings, where template sample is limited, increasing template concentration is not an option.
In these cases where greater sensitivity is necessary, Turbo Primers have demonstrated much promise. When compared to unmodified primers, amplicon formation in a triplex reaction was detected at a hundred-fold lower input of template when using Turbo Primers (Figure 6A), with Turbo Primers efficiently detecting 50 copies of Lambda genomic DNA, where unmodified primers could only detect 5,000 copies. Furthermore, the longer targets appeared to amplify less efficiently than the shorter targets when using unmodified primers, with the longest 962 bp amplicon not forming until 50,000 copies of template were employed. On the other hand, at all template concentrations examined, Turbo Primers amplified all three targets with similar efficiency. Furthermore the use of CleanAmp™Primers improved PCR performance by reducing primer dimer formation (Figure 6B). In summary, efficient amplification by Turbo Primers is less restricted by target size limitations, all three amplicons being formed over a broad range of input template concentrations at increased sensitivity.
To confirm that the range of detection was also reproducible in quantitative real-time PCR, a duplex reaction using Taqman® probe detection was performed. Much like the endpoint experiments, at low template concentrations, the detection of amplification is much more sensitive using CleanAmp™ Turbo Primers. In this duplex reaction, the difference in Cq between unmodified and Turbo Primers increased as template concentration decreased (Figure 7). In some cases, such as the in the L600 target, no Cq is observed at 50 copies for the unmodified primers. Turbo Primers provide earlier detection of successful amplification, whereas amplicon detection with unmodified primers is delayed or not present.
Overall, the use of CleanAmp™ Turbo Primers in multiplex PCR provides several advantages, which include greater amplicon yield and lower primer dimer formation. Turbo Primers provide great flexibility in assay design, as a wide range of primer concentrations produce robust, non-preferential amplification. Furthermore, greater sensitivity is achieved for both endpoint and real time assays, with a 100-fold increase in the limit of detection.
With the advent of microarrays, the need to validate the massive amount of gene expression results has grown significantly. Reverse transcription PCR (RT-PCR) has become the gold standard for validation of microarray gene expression profiles(8,9). The typical RT-PCR reaction consists of a two-step protocol that involves a lower temperature reverse transcription step followed by an elevated temperature PCR step(10). The extra manipulation procedures inherent to a two-step protocol can introduce opportunities for contamination. A one-step RT-PCR protocol provides a streamlined, high-throughput technique that reduces the chances of contamination(11). Another advantage for a one-step protocol is that replicates will repeat both the reverse transcription and the PCR step. However, one-step RT-PCR is not without its own inherent problems. In many cases one-step RT-PCR reactions are not as sensitive as two-step(12,13). The lack of sensitivity is likely a result of reverse transcriptase(14) or DNA polymerase(15) mediated extension of primers to form primer-dimer and/or non-specific products at the less stringent temperatures of reverse transcription. To improve the sensitivity and specificity of RT-PCR, inhibition of such primer extension at lower temperatures is required.
One approach to improving the specificity of one-step RT-PCR is to employ CleanAmp™ Primers. By introducing a CleanAmp™ Primer pair, only the RT primer can elongate during reverse transcription. This reduces lower-temperature, non-specific amplicon formation from extension of PCR primers. At higher temperature, the CleanAmp™ Primers are activated, allowing for greater specificity of primer extension during PCR. CleanAmp™ Primers provide a solution to non-specific amplifications and also enable other more universal RT priming methods for applications such as multiplex one-step RT-PCR.
CleanAmp™ Primers decrease competing PCR primer extension during the RT step with a number of commonly used RT enzymes. To illustrate this, PCR primer sequences were prepared as either a) standard, unmodified primers or b) one of two types of CleanAmp™ Primers (16,17). CleanAmp™ Turbo Primers and slower-releasing CleanAmp™ Precision Primers differ in the rate of temperature-induced formation of the corresponding unmodified primer. The following RT enzymes were tested: SuperScript® II Reverse Transcriptase (Invitrogen) and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) (Invitrogen) (Figure 8), with similar results found for both RT enzymes. The use of unmodified PCR primers resulted in formation of several non-specific amplicons, with the desired amplicon (264 bp) being formed at low relative abundance. Amplicon formation was enriched when CleanAmp™ Turbo Primers were used. In the case of CleanAmp™ Precision Primers, amplicon formation was also enriched to a slightly lesser degree. However, reactions containing Precision Primers displayed improved specificity.
CleanAmp™ Primers can also be utilized with reactions employing either total RNA or poly(A)+ RNA. Experiments have shown that with various RNA tissue sources, one step RT-PCR displayed improved specificity when CleanAmp™ Primers were employed. Furthermore, one-step RT-PCR experiments have been evaluated in Real-Time qRT-PCR using CleanAmp™ Turbo and Precision Primers. The use of CleanAmp™ Primers enhanced the specificity of the reaction compared to the unmodified primers. CleanAmp™ Turbo Primers displayed the most significant increase in amplicon formation. Overall, CleanAmp™ Primers demonstrated marked improvement of sensitivity and specificity in one-step endpoint and real-time RT-PCR protocols.
CleanAmp™ Primers were examined for their ability to support multiplexed one-step RT-PCR. Gene A (264 bp) and gene B (205 bp) were employed in singleplex and duplex reactions (Figure 9). In both singleplex and duplex reactions, unmodified primers produced off-target amplification products. CleanAmp™ Turbo and CleanAmp™ Precision Primers improved the specificity of singleplex reactions, CleanAmp™ Precision Primers providing the highest enrichment in multiplex amplification.
This unique thermolabile modification protects PCR primers from extension during the RT step until thermally activated in the PCR step, thereby reducing non-specific product formation in one-step RT-PCR protocols. Non-Hot Start DNA polymerases, such as Taq DNA polymerase, and standard reverse transcriptases, such as M-MLV reverse transcriptase, are recommended. There is no need to use more expensive modified DNA polymerases or RT enzymes. Furthermore, poly dT18 or random primers can be employed during the cDNA synthesis steps as opposed to a gene-specific primer. This universal priming method allows for multiplex one-step RT-PCR reactions. This is also a key advantage of CleanAmp™ Primers because other one-step RT-PCR kits do not recommend the use of poly dT18 or random primers. In summary, CleanAmp™ Precision Primers are a significant improvement over unmodified primers when used in one-step RT PCR.
In PCR applications such as diagnostics or medical testing, efforts have been directed at reducing the duration of the PCR amplification step, allowing for faster results and greater throughput. However, these faster cycling PCR protocols also have some drawbacks including a decrease in sensitivity and a loss in reproducibility (18). One suggested solution to this decrease in sensitivity is to increase primer concentration.
The main complication encountered with this solution is that an increase in primer concentration often leads to higher off-target amplification, which can increase the false positive rate in real-time experiments, especially with SYBR Green detection (18). Therefore, prevention of off-target amplification at high primer concentration could lead ultimately to an increase in sensitivity when using fast cycling PCR. Herein, the utility of CleanAmp™ Turbo Primers in preventing off-target amplification at high primer concentration using fast cycling PCR protocols will be demonstrated.
Turbo Primers were evaluated for their ability to reduce off-target amplification and to improve the sensitivity of real-time SYBR Green assays using a typical fast cycling protocol (Figure 10). When unmodified primers were employed, interpretation of the amplification plots was complicated by strong amplification of the NTC sample. Although 125 copies of template DNA was accurately detected, the NTC sample amplified before each of the subsequent lower template concentrations. In comparison, the use of CleanAmp™ Turbo Primers allowed for detection down to a single copy. The NTC amplification curve did not cross the threshold line (Cq) during the 40 thermal cycles of the experiment. In addition, when the Cq values were plotted in standard curves (Figure 10B), CleanAmp™ Turbo Primers exhibited greater linearity compared to unmodified primers. This characteristic is most likely related to the suppression of primer dimer formation when CleanAmp™ Turbo Primers are used and demonstrates Turbo Primer's increased specificity over a large range of template concentrations.
Next, CleanAmp™ Turbo Primers were compared with Full Velocity™ SYBR Green QPCR Mastermix (Stratagene/Agilent), a commercially available kit which is formulated specifically for fast cycling PCR. The experiment involved amplification of a 245 base pair target from Lambda genomic DNA. To accurately compare the two products in the same experiment, a 5 minute initial denaturation time necessary for the Full Velocity™ Mastermix was used with these cycling conditions. We found that Turbo Primers had slightly greater sensitivity than the Full Velocity™ Mastermix.
As depicted in Figure 11A, the NTC curve overlapped with the 5 copy sample with Full Velocity™ Mastermix. However, when Turbo Primers were employed, 5 copies of template amplified before the NTC curve. These findings are again likely due to a lower level of off-target amplification by Turbo Primers. When the experimental results were plotted in a standard curve, both approaches displayed good linearity over the range of concentrations evaluated. Although the efficiencies were similar for each technology, reactions that employed Turbo Primers had lower Cq's. This allowed for even greater speed of amplicon detection in a fast cycling protocol.
In summation, CleanAmp™ Primers allow for greater control of primer hybridization and extension during PCR. Over a broad range of applications CleanAmp™ Primers reduce or eliminate off-target amplification. Greater amplicon yield is also achieved, due to improvement in specificity and sensitivity. It has been demonstrated that CleanAmp™ Primers outperform other technologies in multiple applications.
Which CleanAmp™ Primer is best for my application?
|Turbo Primers||Precision Primers|
|Fast cycling||Standard cycling|
|Multiplex PCR||One-step reverse-transcription PCR|
|Improves amplicon||Improved specificity and yield limit of detection|
|Reduces mis-priming/primer dimer formation||Greatest reduction in mis-priming/primer dimer formation|
Glen Research is delighted to be able to support the CleanAmp™ technology by making the CleanAmp™ monomers available for sale and by supporting the synthesis of Turbo and Precision Primers. This is being done under license from TriLink BioTechnologies, Inc.. The following article describes the synthesis and purification of CleanAmp™ Primers.