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Getting Things Backwards to Prevent Primer Dimers

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Getting Things Backwards to Prevent Primer Dimers The Journal of Molecular Diagnostics, Vol. 16, No. 2, March 2014 jmd.amjpathol.org See related article on page 163. COMMENTARY Getting Things Backwards to Prevent Primer Dimers Mark A. Poritz and Kirk M. Ririe From BioFire Diagnostics, Inc., Salt Lake City, Utah Enzymatic synthesis of DNA occurs in the 50 to 30 direction by compete with the formation of the target amplicon. Primer addition of monomers to the 30 end of the new strand. In dimers are believed to be formed by the chance base pairing contrast, standard chemical synthesis of nucleic acid occurs in and extension of each primer, using the other primer as a the opposite direction, 30 to 50, by the addition of nucleoside template.7,8 Presumably, the high concentration of the 30-O-phosphoramidites to the 50 end of the oligonucleotide. primers in the PCR drives the formation of an initial ternary However, nucleosides containing 50-O- phosphoramidites are complex of Taq DNA polymerase with the two oligonucle- commercially available and can be used in conventional otides, but this event has never been directly observed. Given automated DNA synthesizers. Such reverse phosphoramidites the great effort that has been made to minimize their extend the growing chain in the 50 to 30 direction.1 Reverse and appearance, it is surprising how little work has gone into standard phosphoramidites also have been combined in the characterizing primer dimers at the molecular level. Brownie same synthesis to make unusual DNA molecules with alter- et al9 cloned and sequenced primer dimers; when they nating 30 to 30 and 50 to 50 phosphodiester linkages.2 aligned these sequences with the primers that generated them, Until now, the general utility of reverse phosphoramidites they observed that a few had zero overlap between the has not been clear and their use has been quite limited. This primers, most had between 2 and 10 nucleotides of untem- is likely to change with the publication of an article by plated sequence inserted between the primers, and some Satterfield3 in this issue of The Journal of Molecular required that the primers bind to the primer dimer with 30 Diagnostics. Satterfield3 used both standard and reverse mismatches. Clearly, there is much we do not understand phosphoramidites to synthesize novel oligonucleotides that about the formation of primer dimers. contained two 30 ends, and no 50 end. When used as primers Over the quarter of a century since PCR was invented in PCR, these molecules have the striking ability to suppress numerous methods have been developed to prevent the primer dimer amplification. This is an important technical occurrence of primer dimers. Computer algorithms weed out contribution to nucleic acid amplification technology that primer sets that can base pair and extend into primer also hints that oligonucleotides synthesized with different dimers.10 Physical methods have been used to prevent the polarities will find more general use in molecular biology reaction from starting before the components are brought to and molecular diagnostics. the annealing temperature.7,11 Almost every molecule in the reaction, from the enzyme to the primers to the magnesium Primer Dimers ions, has been modified or sequestered so as to be unavailable until the reaction is ready to begin (see references in the 3 12 The development of highly sensitive, highly multiplexed articles by Satterfield and Lebedev et al ). These methods assays for detecting infectious agents4,5 or cancer genotyp- work to varying degrees but they all share one important ing6 is made possible by the high signal-to-noise ratio of defectdthey do not prevent the amplification of primer basic PCR technology, which in turn is dependent on the exponential nature of specific target amplification. Pushing Accepted for publication January 9, 2014. PCR to even greater sensitivity can be difficult because of the Disclosures: M.A.P. and K.M.R. are employed by BioFire Diagnostics, Inc. formation of two side products: primer dimers and off-target Address correspondence to Mark A. Poritz, Ph.D., BioFire Diagnostics, amplicons (the result of mispriming events on genomic Inc., 390 Wakara Way, Salt Lake City, UT 84108. E-mail: mark.poritz@ nucleic acid). Both of these reactions are also exponential and biofiredx.com Copyright ª 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jmoldx.2014.01.001 Poritz and Ririe Figure 1 Cooperative primers amplify template, but not primer dimers. A and B: Cooperative primers (red lines and green lines) are shown during the PCR amplification of a template (yellow lines and blue lines). The two forms of cooperative primers consist of two DNA sequences joined together either 50 to 50 (A)or50 to 30 (B) through a poly- ethylene glycol linker (shown as a dotted arc).3 The 30 ends of the cooperative primers are indicated by arrowheads. The vertical line on the 30 end of the capture sequence (A) in- dicates that it is chemically blocked from being extended. The initiation (I) and propagation (P) steps for primer dimers are shown for cooperative (C) and conventional (D) primers. Satterfield’s3 data indicate that cooperative primers block the propagation step for primer dimers (C, red cross).3 For brevity, only the bottom strand is included in the DNA syn- thesis steps depicted here. dimers once they have been formed for the simple reason that Satterfield3 showed that both forms of cooperative primers a primer dimer is a perfect substrate for the primers that can function in a variety of different PCRs. Compared with generated it (Figure 1). similar reactions using conventional primers, those run with cooperative primers are a million-fold more resistant to in- hibition by synthetic primer dimers spiked into the reaction. Cooperative Primers As expected, an antibody-mediated hot start did not suppress the inhibition caused by primer dimers spiked into a con- Satterfield’s3 attack on the primer dimer problem is based on ventional PCR. novel primer designs that prevent the amplification of existing These data show that hot start methods applied to con- primer dimers. It builds on previous work13 with modified ventional primers can suppress only the initiation, but not the molecular beacons (termed tentacle probes) that separate the propagation, of primer dimers (Figure 1D). In contrast, recognition of an amplicon into two regions: one for capture cooperative primers prevent the propagation of primer dimers and one for recognition of a polymorphism. This principle now (Figure 1C). The primer dimer sequence data described has been applied to the binding of a PCR primer to its target: a earlier9 suggests that the initial step for primer dimer for- capture sequence provides most of the specificity of target mation by conventional primers is not necessarily dependent recognition but cannot be extended, whereas an attached on base pairing. It seems likely that the initiation of primer primer sequence can extend to form the amplicon. These oli- dimers also is not blocked by cooperative primers gonucleotides are termed cooperative primers in recognition of (Figure 1C). However, this is not essential because the the two binding events. In this respect they are similar to the propagation step is inhibited. It is likely that the low melting previously described dual priming oligonucleotides14;how- temperatures of the short, extendable section of the primers ever, cooperative primers have a novel topologic feature that prevents them from hybridizing to the primer dimer at the makes them uniquedthe capture oligonucleotide is down- annealing temperature of the reaction. This model predicts stream (30) of the primer that will be extended. This arrange- that primer dimers will start to be formed as the length of the ment ensures a high degree of sequence specificity toward the extendable primer increases but the true test of this theory correct template in every cycle of PCR (Figure 1, A and B) may require trapping the rare ternary complex of Taq and while minimizing the length of oligonucleotide that can hy- primers in a form that can be studied. bridize to a primer dimer (Figure 1C). Cooperative primers come in two forms. In one form the Future Steps oligonucleotide is made by conventional DNA synthesis with a series of polyethylene glycol linkers separating the Satterfield3 has shown that singleplex PCR reactions using capture and primer sequences (Figure 1B) [in this case a cooperative primers are highly resistant to inhibition by fluorescent dye and quencher (not shown) are placed on the externally added primer dimers. The next step would be to capture sequence for real-time PCR applications]. The sec- show that cooperative primers provide the same benefitto ond form of cooperative primer places the capture sequence multiplex PCR reactions. Cooperative primer design is going (with a blocked 30 end) and the primer sequence in a head- to be more complicated than that for standard primers to-head configuration (50 to 50, separated by polyethylene because two different binding sites (the capture and primer glycol linkers) (Figure 1A). This is achieved by synthesizing sequences) need to be identified and optimized. Satterfield3 part of the DNA molecule using the aforementioned reverse provides suggestions, based on thermodynamic arguments phosphoramidites. and on the initial PCR data, for what the respective 160 jmd.amjpathol.org - The Journal of Molecular Diagnostics Preventing Primer Dimer Amplification oligonucleotide melting temperatures need to be, but the specific and nonspecific amplicons generated in a multiplex multiplex PCR design imposes the additional requirement PCR reaction without the biases introduced by cloning.9 As that all primers perform well at a single temperature. the costs of next-generation sequencing continue to decrease Combining this constraint with the sequence requirements of it is possible that such analysis will become a standard part of the amplicons may best be performed empirically.
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