Polymerase Chain Reaction (PCR) is a nucleic acid amplification technique that is used in various applications, such as diagnostics, forensics, agriculture, and basic research. PCR uses a polymerase to synthesize many, many copies of a target sequence over repeated cycles of denaturing and renaturing the sample (Figure 1). The PCR polymerase relies on DNA primers and deoxynucleotide triphosphates (dNTPs) to recognize the target nucleic acid sequence and generate the complementary strand to the template, respectively. PCR primer design is where Glen Research comes into play.
Figure 1. Illustration of PCR
PCR primers must be able to selectively recognize the target sequence to obtain optimal amplification. Obviously, the primer must have the right base composition to hybridize to the 3′ region of the target for polymerase extension. In addition to the correct sequence, several base modifications have been evaluated in their ability to improve PCR efficiency.
When it comes to PCR modifications, some obvious backbones come to mind. Anything that can enhance hybridization and melting temperatures (Tm) has been used to enhance PCR efficiency. These include Locked Analog, 2′-OMe, and 2′-F. However, for the scope of this article, we want to focus on some of our less well-known modifications for PCR primers. It’s worth pointing out that these modifications can also apply to quantitative PCR (qPCR) probes, which use the same strategy but contain a reporter molecule.
Self-Avoiding Molecular Recognition Systems (SAMRS)
Self-Avoiding Molecular Recognition Systems, or SAMRS, utilize modified bases that form more stable base pairs with the natural complement than with their SAMRS complement.1 The SAMRS bases analogs include 2-Aminopurine (A*), N4-Et-dC (C*), dI (G*), and 2-Thio-dT (T*) (Figure 2). The A analog, 2-Aminopurine, preferentially binds natural thymidine over 2-Thio-dT due to one less hydrogen bond (Figure 3). Similarly, C* binds stronger to G than G* and G* binds stronger to C than C*, based on melting temperatures.1
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Figure 2. SAMRS base phosphoramidites | Figure 3. Base pairing motifs |
This technology enables PCR primers using SAMRS bases to avoid undesired intra- and intermolecular hybridization between primers. This is particularly beneficial in multiplexed PCR techniques.1, 2 Primer dimer amplification is often more efficient than the desired target, so primer-primer interactions should be avoided to prevent reagent consumption. Preventing the formation of primer dimers can improve discrimination between single nucleotide polymorphisms (SNPs).2, 3
PCR primers are not made fully with SAMRS modifications. The amount of SAMRS in each primer must be optimized to enhance target binding and discourage primer dimer formation. For this system to work best, primers should be at least 20 nucleotides
long and contain 1-3 SAMRS modifications.2 Generally, the least destabilizing monomer is T*, followed by A* and C*, and lastly G*. G-rich primers have lower amplification efficiency than other sequences. Ideally, the 3′-most base should be natural DNA.
Notably, SAMRS technology has also been used in isothermal amplification techniques, including loop-mediated isothermal amplification (LAMP) and recombinase-based isothermal amplification.4, 5
Other Base Modifications
In addition to SAMRS, other base modifications have been used to improve PCR efficiency. A PCR method, termed Snake, used forward primers with a 5′-flap bearing base modification that is recognized by the 5′-nuclease activity of the popular Taq DNA polymerase.6 The 5′-flap sequence enables a stem-loop structure in the Snake PCR amplicon, which improved signal productivity and SNP discrimination. Certain positions of the 5′-flap sequence were substituted with 2-Amino-dA and/or 5-propynyl dU (pdU) to enhance binding (Figure 4). Each 2-Amino-dA increases Tm by 3.0 °C while a pdU addition yields a +1.7 °C per substitution.7 Similar to SAMRS, the amount of these modifications must be optimized to prevent too stable complexes from forming and some destabilizing modifications (dI and dU) may be added to regulate this.
Figure 4. Base modifications for Snake primer 5′-flap
Non-base Modifications
Lastly, the addition of a simple 5′-thiol to the end of the PCR has been shown to enhance PCR sensitivity and yield.8 The proposed mechanism for this enhancement is due to the interaction between the primer and the PCR enzymes, namely the polymerase.
The major benefit of this approach is that it does not need significant optimization to identify how many and which base modifications are needed to improve PCR outcomes. This technique required contaminant-free reactions as the presence of other proteins in the sample inhibited PCR with thiol-modified primers.
Technique | Product | Catalog No. |
SAMRS | ||
Snake | ||
Thiol |
References
