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. A large the development and optimization of a multiplex PCR. number of primers will have to be tested but this may be Cooperative primers will not solve the other vexing balanced by two benefits that cooperative primers offer. First, problem of PCRdamplicon contamination. However, it can if cooperative primers are as resistant to primer dimer be easier to detect false-positive than false-negative results amplification as shown here, then the iterations of primer in clinical samples. As Satterfield3 notes, it is entirely design (in silico and in vitro)15e17 that are required to mini- possible that primer dimers have caused unrecognized mize primer dimers will not be required. Second, similar to sporadic false-negative results (especially when detecting the dual priming oligonucleotides mentioned earlier,14 rare pathogens). Cooperative primers will add a power tool cooperative primers bind to two separate sequences. This to the PCR toolkit that should prevent this occurrence. will be a major advantage when designing multiplex PCRs to detect pathogens. Viral and bacterial genomes contain blocks of conserved sequence separated by regions of high diversity. References Cooperative primers, with their inverted design and poly- 0 fi ethylene glycol linkers, actually may function better, because 1. Claeboe CD, Gao R, Hecht SM: 3 -modi ed oligonucleotides by reverse DNA synthesis. Nucleic Acids Res 2003, 31:5685e5691 of reduced steric hindrance, when the capture and primer 2. Koga M, Geyer SJ, Regan JB, Beaucage SL: The synthesis of alter- binding sites are separated by tens to hundreds of nucleotides. nating alpha,beta-oligodeoxyribonucleotides with alternating (30–>30)- Methods for preventing primer dimer formation in PCR and (50–>50)-internucleotic linkages as potential therapeutic agents. also may apply to any of the numerous isothermal amplifi- Nucleic Acids Symp Ser 1993, 29:3e4 fi cation strategies that are competing with PCR for use in the 3. Satter eld BC: Cooperative primers: a 2.5 million-fold improvement in 18 the reduction of nonspecific amplification. J Mol Diagn 2013, 16: laboratory and the clinic. Unfortunately, primer design 162e172 for the widely used loop-mediated isothermal amplification 4. Mahony JB, Petrich A, Smieja M: Molecular diagnosis of respiratory (LAMP) reaction is complicated enough to require custom virus infections. Crit Rev Clin Lab Sci 2011, 48:217e249 software19; combining this with the complexity of coop- 5. Navidad JF, Griswold DJ, Gradus MS, Bhattacharyya S: Evaluation of fi erative primers will take some effort but the benefits may Luminex xTAG gastrointestinal pathogen analyte-speci c reagents for fi high-throughput, simultaneous detection of bacteria, viruses, and par- be worth the added dif culty. This technology also may asites of clinical and public health importance. J Clin Microbiol 2013, have utility for next-generation sequencing protocols that 51:3018e3024 use a PCR step to prepare the library to be sequenced. 6. Beadling C, Neff TL, Heinrich MC, Rhodes K, Thornton M, Reactions that generate fewer primer dimers should require Leamon J, Andersen M, Corless CL: Combining highly multiplexed less clean-up. PCR with semiconductor-based sequencing for rapid cancer genotyp- ing. J Mol Diagn 2013, 15:171e176 7. Chou Q, Russell M, Birch DE, Raymond J, Bloch W: Prevention of pre-PCR mis-priming and primer dimerization improves low-copy- Significance number amplifications. Nucleic Acids Res 1992, 20:1717e1723 8. Hsu JT, Das S, Mohapatra S: Polymerase chain reaction engineering. e Several novel ideas were combined to develop cooperative Biotechnol Bioeng 1997, 55:359 366 9. Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, primers and they will have uses beyond those presented. In a Newton C, Little S: The elimination of primer-dimer accumulation in few years, standard PCR might be performed with a single PCR. Nucleic Acids Res 1997, 25:3235e3241 oligonucleotide that combines the forward and reverse 10. Alvarez-Fernandez R: Explanatory chapter: PCR primer design. primers into one molecule. The resulting amplicons could Methods Enzymol 2013, 529:1e21 have interesting melting properties that can be exploited for 11. Poritz MA, Blaschke AJ, Byington CL, Meyers L, Nilsson K, Jones DE, Thatcher SA, Robbins T, Lingenfelter B, Amiott E, better detection or differentiation. Primers with two 30 ends 20 Herbener A, Daly J, Dobrowolski SF, Teng DH, Ririe KM: FilmArray, also may find utility in DNA origami applications and this an automated nested multiplex PCR system for multi-pathogen may lead in turn to better tools for diagnostics (see Hartman detection: development and application to respiratory tract infection. et al21 for a similar idea). PLoS One 2011, 6:e26047 The demand for deep multiplexing amplification tech- 12. Lebedev AV, Paul N, Yee J, Timoshchuk VA, Shum J, Miyagi K, Kellum J, Hogrefe RI, Zon G: Hot start PCR with heat-activatable niques is only going to increase. Although strategies such as primers: a novel approach for improved PCR performance. Nucleic cooperative primers will facilitate such protocols, they also Acids Res 2008, 36:e131 highlight the complexity of these reactions at the molecular 13. Satterfield BC, West JA, Caplan MR: Tentacle probes: eliminating level. Even as we reduce primer dimers, we need to better false positives without sacrificing sensitivity. Nucleic Acids Res 2007, understand the sources of nonspecific amplification. The 35:e76 14. Chun JY, Kim KJ, Hwang IT, Kim YJ, Lee DH, Lee IK, Kim JK: Dual community would be well served by efforts to better char- priming oligonucleotide system for the multiplex detection of respi- acterize all of the products of a multiplex amplification. ratory viruses and SNP genotyping of CYP2C19 gene. Nucleic Acids Next-generation sequencing can characterize, in depth, the Res 2007, 35:e40

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