Designing reproducible PCR assays involves optimizing multiple moving targets, from standardizing each component in sometimes minute reaction volumes to planning ahead to ensure long-term and secure storage of reagents, samples, and PCR products.
Appreciating the intricacies and common challenges involved in the development and optimization of PCR-based diagnostic tests will help researchers overcome such roadblocks using the latest solutions available. Here we discuss some of the common challenges that researchers may encounter when designing a PCR-based molecular diagnostic assay and some potential solutions.
Polymerases and primers
One of the first challenges one encounters in designing a PCR reaction is choosing the optimal polymerase for the application. Weighing your needs in the context of the characteristic of the biological sample, the length of the sequence to be amplified, and desired sequence accuracy of the amplified product is critical.
Gerald Hunter, PhD, field application scientist at Fortis Life Sciences, who has extensive experience in optimizing PCR assays says, “Different DNA polymerases have different specificities, enzymatic functions, error rates (fidelities), and tolerance to PCR inhibitors.”
Designing primers that anneal to DNA templates at an intended temperature with high specificity and optimizing the composition of the reaction mix poses the next challenges.
“Success in PCR is based on the ability of your reaction to maintain a low ratio of nonspecific primer binding and therefore the annealing temperature becomes a critical factor that must be optimized,” says Hunter. “In addition, the PCR buffer components—pH, salt, Mg2+, primer, and enzyme concentration, which all play a factor in the performance of the reaction—must be considered and may require optimization.”
Nonspecific amplification generally occurs when primers bind to sequences they are not designed for. This results in unintended products.
“Primer design is both an art and a science. Not only is the right primer concentration important, but also having the right primer sequence,” says Hunter. “Luckily, there are many free, online primer design software tools that can be used for PCR primer design, but don’t be surprised if you find yourself having to test a handful of primer pair sets before you find the right combination.”
One way to avoid nonspecific amplification is to prepare your reaction mix on ice until you’re ready to insert your PCR tube or strip into the thermal cycler. The low temperature reduces the activity of the DNA polymerase decreasing the likelihood of primers binding imprecisely. Despite this precaution, undesired products may still be synthesized.
An additional solution to counter nonspecific amplification is to use a hot-start DNA polymerase. The hot-start DNA polymerase is chemically modified to be inactive at room temperature and prevents nonspecific amplification. Nucleotides are only strung together by the modified enzyme once a heat activation step has occurred. This allows the assembly of the PCR reaction at room temperature, without the possibility of spurious products and the formation of primer dimers. An added advantage of the hot-start PCR is that reaction cycling conditions with no heat activation can be used as a negative control.
Multiplexing is the ability to detect a range of conditions or factors in a single test, as opposed to conducting individual tests for each parameter.1 It benefits throughput, lowers costs for tests, and improves the efficiency of patient care.
Multiplexing is often challenged by primer design. Primers for multiplex PCRs must be highly selective for target sequencing with consistent melting temperatures that produce amplicons of uniform length. This requires careful computation when designing primers. If designed improperly, primers form homo- or hetero-dimers, or amplify off-target sequences.
“Multiplex PCR typically requires an additional oligo called a probe. To multiplex two or more assays, these probes must be distinguishable from each other and detectable simultaneously,” says Hunter.
Solutions for multiplexing include the use of hot-start polymerases and primer design algorithms. The algorithms limit primer dimerization due to complementarity and off-target amplification through sequence specificity.
Hunter adds, “Primer design algorithm(s) used must account for sequence specificity and limit both off-target and primer dimer effects and the DNA polymerase used for amplification must be stringently hot-start modified to ensure that all primers are completely melted and specifically annealed to their target sequence before the polymerase becomes active. Optimally, this polymerase will be enabled for fast thermocycling conditions and have extremely low background activity at ambient temperatures.”
Sample and carryover contaminants
Environmental and tissue samples such as soil and saliva often contain components that inhibit PCR reactions by binding with polymerases, enzyme cofactors, genomic DNA, or amplified single or double-stranded DNA or RNA. Contaminants can reduce DNA isolation, polymerase activity, and analytical and diagnostic sensitivity.
For example, PCR inhibitors are frequently found in stool samples. These may originate from dietary components and lead to false negative PCR results. In addition, heme in blood samples and collagen in tissue samples can also inhibit PCR. The addition of BSA (bovine serum albumin) to the reaction mastermix and the use of inhibition-resistant polymerases are effective methods for eliminating the inhibitory effect of these compounds.2
PCR is sensitive enough to detect a single copy gene from a single cell. This extreme sensitivity poses a problem as it can also detect contaminating trace DNA or RNA fragments from a previous reaction or extraneous sources.
“Many labs are now considering using uracil-DNA glycosylases (UDGs) in their PCR workflows to prevent carryover contamination,” says Hunter.
UDGs are highly conserved DNA repair enzymes. Its function is to remove uracil from DNA. Uracil is normally found in RNA not DNA. When UDGs and dUTPs are added to the PCR reaction, UDGs can selectively degrade DNA from earlier PCR reactions that contain dUTP instead of dTTP. This step occurs at 37°C before hot-start activation of the polymerase. The presence of uracil instead of thymine does not affect the electrophoretic mobility or sequencing of the PCR products, and the activity UDG is destroyed during hot-start activation.
“Another tip would be to use inhibitor-tolerant PCR master mixes for difficult or dirty sample types,” says Hunter.
When time is of the essence, such as when viral RNA must be detected to confirm infections, the two-step process of reverse transcription followed by amplification can be compressed into a one-step RT-qPCR. This allows both reactions to be carried out in a single tube in a single step, reducing chances of contamination as well as hands-on time.
In addition, slow reaction kinetics in end-point PCRs and qPCRs may result in a workflow that takes hours. Advances in qPCR dyes, probes, and thermocycling technologies, such as faster heating and cooling mechanisms, and advances in polymerase and buffers have greatly reduced the total time for PCR and made molecular diagnostics at POC (point of care) feasible.
PCR products and reagents are normally transported on dry ice, but this entails the risk of the dry ice evaporating before packages reach their destination. Long-term ambient storage of nucleic acid samples, PCR reagents, and amplified products is preferentially achieved through lyophilization and air-drying solutions that preclude the need for storage in dry ice during transportation and cold storage in the laboratory.
“We are seeing a lot of point-of-care companies expanding their use of lyophilization, specifically lyophilized beads, for enzymes and PCR master mixes. Lyophilized beads are more reliable, easier to handle, and require no aliquoting and can be used and stored at ambient temperature which makes them ideal in point-of-care settings,” says Hunter.
Lyophilization, freeze drying, or sublimation provides an alternative solution for long-term shelf life at ambient temperatures by removing water from the product without heating the product excessively. The process involves freezing and placing the product in a vacuum and can be carried out in a single tube. When the water is removed, it is replaced by an excipient such as trehalose, dextran, or lactose. The use of lyophilization beads aids in the standardization of the process.
Optimizing a PCR assay involves elements of science, art, trial and error, and patience. Knowing what might go wrong and what you could do in such situations, often tips the scale from failure to success.
- Khodakov D, Li J, Zhang JX, Zhang DY. Highly multiplexed rapid DNA detection with single-nucleotide specificity via convective PCR in a portable device. Nat Biomed Eng. 2021 Jul;5(7):702-712. PMID: 34211146.
- Oikarinen S, Tauriainen S, Viskari H, Simell O, Knip M, Virtanen S, Hyöty H. PCR inhibition in stool samples in relation to age of infants. J Clin Virol. 2009 Mar;44(3):211-4. Epub 2009 Feb 3. PMID: 19196549.