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Getting Things Backwards to Prevent Primer Dimers

2014; Elsevier BV; Volume: 16; Issue: 2 Linguagem: Inglês

10.1016/j.jmoldx.2014.01.001

1943-7811

Mark A. Poritz, Kirk M. Ririe,

RNA and protein synthesis mechanisms

Enzymatic synthesis of DNA occurs in the 5′ to 3′ direction by addition of monomers to the 3′ end of the new strand. In contrast, standard chemical synthesis of nucleic acid occurs in the opposite direction, 3′ to 5′, by the addition of nucleoside 3′-O-phosphoramidites to the 5′ end of the oligonucleotide. However, nucleosides containing 5′-O- phosphoramidites are commercially available and can be used in conventional automated DNA synthesizers. Such reverse phosphoramidites extend the growing chain in the 5′ to 3′ direction.1Claeboe C.D. Gao R. Hecht S.M. 3′-modified oligonucleotides by reverse DNA synthesis.Nucleic Acids Res. 2003; 31: 5685-5691Crossref PubMed Scopus (14) Google Scholar Reverse and standard phosphoramidites also have been combined in the same synthesis to make unusual DNA molecules with alternating 3′ to 3′ and 5′ to 5′ phosphodiester linkages.2Koga M. Geyer S.J. Regan J.B. Beaucage S.L. The synthesis of alternating alpha,beta-oligodeoxyribonucleotides with alternating (3′-->3′)- and (5′-->5′)-internucleotic linkages as potential therapeutic agents.Nucleic Acids Symp Ser. 1993; 29: 3-4PubMed Google Scholar Until now, the general utility of reverse phosphoramidites has not been clear and their use has been quite limited. This is likely to change with the publication of an article by Satterfield3Satterfield B.C. Cooperative primers: a 2.5 million-fold improvement in the reduction of nonspecific amplification.J Mol Diagn. 2013; 16: 162-172Google Scholar in this issue of The Journal of Molecular Diagnostics. Satterfield3Satterfield B.C. Cooperative primers: a 2.5 million-fold improvement in the reduction of nonspecific amplification.J Mol Diagn. 2013; 16: 162-172Google Scholar used both standard and reverse phosphoramidites to synthesize novel oligonucleotides that contained two 3′ ends, and no 5′ end. When used as primers in PCR, these molecules have the striking ability to suppress primer dimer amplification. This is an important technical contribution to nucleic acid amplification technology that also hints that oligonucleotides synthesized with different polarities will find more general use in molecular biology and molecular diagnostics. The development of highly sensitive, highly multiplexed assays for detecting infectious agents4Mahony J.B. Petrich A. Smieja M. Molecular diagnosis of respiratory virus infections.Crit Rev Clin Lab Sci. 2011; 48: 217-249Crossref PubMed Scopus (140) Google Scholar, 5Navidad J.F. Griswold D.J. Gradus M.S. Bhattacharyya S. Evaluation of Luminex xTAG gastrointestinal pathogen analyte-specific reagents for high-throughput, simultaneous detection of bacteria, viruses, and parasites of clinical and public health importance.J Clin Microbiol. 2013; 51: 3018-3024Crossref PubMed Scopus (127) Google Scholar or cancer genotyping6Beadling C. Neff T.L. Heinrich M.C. Rhodes K. Thornton M. Leamon J. Andersen M. Corless C.L. Combining highly multiplexed PCR with semiconductor-based sequencing for rapid cancer genotyping.J Mol Diagn. 2013; 15: 171-176Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar is made possible by the high signal-to-noise ratio of basic PCR technology, which in turn is dependent on the exponential nature of specific target amplification. Pushing PCR to even greater sensitivity can be difficult because of the formation of two side products: primer dimers and off-target amplicons (the result of mispriming events on genomic nucleic acid). Both of these reactions are also exponential and compete with the formation of the target amplicon. Primer dimers are believed to be formed by the chance base pairing and extension of each primer, using the other primer as a template.7Chou Q. Russell M. Birch D.E. Raymond J. Bloch W. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.Nucleic Acids Res. 1992; 20: 1717-1723Crossref PubMed Scopus (588) Google Scholar, 8Hsu J.T. Das S. Mohapatra S. Polymerase chain reaction engineering.Biotechnol Bioeng. 1997; 55: 359-366Crossref PubMed Scopus (21) Google Scholar Presumably, the high concentration of the primers in the PCR drives the formation of an initial ternary complex of Taq DNA polymerase with the two oligonucleotides, but this event has never been directly observed. Given the great effort that has been made to minimize their appearance, it is surprising how little work has gone into characterizing primer dimers at the molecular level. Brownie et al9Brownie J. Shawcross S. Theaker J. Whitcombe D. Ferrie R. Newton C. Little S. The elimination of primer-dimer accumulation in PCR.Nucleic Acids Res. 1997; 25: 3235-3241Crossref PubMed Scopus (263) Google Scholar cloned and sequenced primer dimers; when they aligned these sequences with the primers that generated them, they observed that a few had zero overlap between the primers, most had between 2 and 10 nucleotides of untemplated sequence inserted between the primers, and some required that the primers bind to the primer dimer with 3′ mismatches. Clearly, there is much we do not understand about the formation of primer dimers. Over the quarter of a century since PCR was invented numerous methods have been developed to prevent the occurrence of primer dimers. Computer algorithms weed out primer sets that can base pair and extend into primer dimers.10Alvarez-Fernandez R. Explanatory chapter: PCR primer design.Methods Enzymol. 2013; 529: 1-21Crossref PubMed Scopus (14) Google Scholar Physical methods have been used to prevent the reaction from starting before the components are brought to the annealing temperature.7Chou Q. Russell M. Birch D.E. Raymond J. Bloch W. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.Nucleic Acids Res. 1992; 20: 1717-1723Crossref PubMed Scopus (588) Google Scholar, 11Poritz M.A. Blaschke A.J. Byington C.L. Meyers L. Nilsson K. Jones D.E. Thatcher S.A. Robbins T. Lingenfelter B. Amiott E. Herbener A. Daly J. Dobrowolski S.F. Teng D.H. Ririe K.M. FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection.PLoS One. 2011; 6: e26047Crossref PubMed Scopus (284) Google Scholar Almost every molecule in the reaction, from the enzyme to the primers to the magnesium ions, has been modified or sequestered so as to be unavailable until the reaction is ready to begin (see references in the articles by Satterfield3Satterfield B.C. Cooperative primers: a 2.5 million-fold improvement in the reduction of nonspecific amplification.J Mol Diagn. 2013; 16: 162-172Google Scholar and Lebedev et al12Lebedev A.V. Paul N. Yee J. Timoshchuk V.A. Shum J. Miyagi K. Kellum J. Hogrefe R.I. Zon G. Hot start PCR with heat-activatable primers: a novel approach for improved PCR performance.Nucleic Acids Res. 2008; 36: e131Crossref PubMed Scopus (42) Google Scholar). These methods work to varying degrees but they all share one important defect—they do not prevent the amplification of primer dimers once they have been formed for the simple reason that a primer dimer is a perfect substrate for the primers that generated it (Figure 1). Satterfield's3Satterfield B.C. Cooperative primers: a 2.5 million-fold improvement in the reduction of nonspecific amplification.J Mol Diagn. 2013; 16: 162-172Google Scholar attack on the primer dimer problem is based on novel primer designs that prevent the amplification of existing primer dimers. It builds on previous work13Satterfield B.C. West J.A. Caplan M.R. Tentacle probes: eliminating false positives without sacrificing sensitivity.Nucleic Acids Res. 2007; 35: e76Crossref PubMed Scopus (18) Google Scholar with modified molecular beacons (termed tentacle probes) that separate the recognition of an amplicon into two regions: one for capture and one for recognition of a polymorphism. This principle now has been applied to the binding of a PCR primer to its target: a capture sequence provides most of the specificity of target recognition but cannot be extended, whereas an attached primer sequence can extend to form the amplicon. These oligonucleotides are termed cooperative primers in recognition of the two binding events. In this respect they are similar to the previously described dual priming oligonucleotides14Chun J.Y. Kim K.J. Hwang I.T. Kim Y.J. Lee D.H. Lee I.K. Kim J.K. Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene.Nucleic Acids Res. 2007; 35: e40Crossref PubMed Scopus (163) Google Scholar; however, cooperative primers have a novel topologic feature that makes them unique—the capture oligonucleotide is downstream (3′) of the primer that will be extended. This arrangement ensures a high degree of sequence specificity toward the correct template in every cycle of PCR (Figure 1, A and B) while minimizing the length of oligonucleotide that can hybridize to a primer dimer (Figure 1C). Cooperative primers come in two forms. In one form the oligonucleotide is made by conventional DNA synthesis with a series of polyethylene glycol linkers separating the capture and primer sequences (Figure 1B) [in this case a fluorescent dye and quencher (not shown) are placed on the capture sequence for real-time PCR applications]. The second form of cooperative primer places the capture sequence (with a blocked 3′ end) and the primer sequence in a head-to-head configuration (5′ to 5′, separated by polyethylene glycol linkers) (Figure 1A). This is achieved by synthesizing part of the DNA molecule using the aforementioned reverse phosphoramidites. Satterfield3Satterfield B.C. Cooperative primers: a 2.5 million-fold improvement in the reduction of nonspecific amplification.J Mol Diagn. 2013; 16: 162-172Google Scholar showed that both forms of cooperative primers can function in a variety of different PCRs. Compared with similar reactions using conventional primers, those run with cooperative primers are a million-fold more resistant to inhibition by synthetic primer dimers spiked into the reaction. As expected, an antibody-mediated hot start did not suppress the inhibition caused by primer dimers spiked into a conventional PCR. These data show that hot start methods applied to conventional primers can suppress only the initiation, but not the propagation, of primer dimers (Figure 1D). In contrast, cooperative primers prevent the propagation of primer dimers (Figure 1C). The primer dimer sequence data described earlier9Brownie J. Shawcross S. Theaker J. Whitcombe D. Ferrie R. Newton C. Little S. The elimination of primer-dimer accumulation in PCR.Nucleic Acids Res. 1997; 25: 3235-3241Crossref PubMed Scopus (263) Google Scholar suggests that the initial step for primer dimer formation by conventional primers is not necessarily dependent on base pairing. It seems likely that the initiation of primer dimers also is not blocked by cooperative primers (Figure 1C). However, this is not essential because the propagation step is inhibited. It is likely that the low melting temperatures of the short, extendable section of the primers prevents them from hybridizing to the primer dimer at the annealing temperature of the reaction. This model predicts that primer dimers will start to be formed as the length of the extendable primer increases but the true test of this theory may require trapping the rare ternary complex of Taq and primers in a form that can be studied. Satterfield3Satterfield B.C. Cooperative primers: a 2.5 million-fold improvement in the reduction of nonspecific amplification.J Mol Diagn. 2013; 16: 162-172Google Scholar has shown that singleplex PCR reactions using cooperative primers are highly resistant to inhibition by externally added primer dimers. The next step would be to show that cooperative primers provide the same benefit to multiplex PCR reactions. Cooperative primer design is going to be more complicated than that for standard primers because two different binding sites (the capture and primer sequences) need to be identified and optimized. Satterfield3Satterfield B.C. Cooperative primers: a 2.5 million-fold improvement in the reduction of nonspecific amplification.J Mol Diagn. 2013; 16: 162-172Google Scholar provides suggestions, based on thermodynamic arguments and on the initial PCR data, for what the respective oligonucleotide melting temperatures need to be, but the multiplex PCR design imposes the additional requirement that all primers perform well at a single temperature. Combining this constraint with the sequence requirements of the amplicons may best be performed empirically. A large number of primers will have to be tested but this may be balanced by two benefits that cooperative primers offer. First, if cooperative primers are as resistant to primer dimer amplification as shown here, then the iterations of primer design (in silico and in vitro)15Camacho J.L. Torres E.M. Cadena C. Prieto J. Prieto L.L. Torregroza D.A. The use of factorial design, image analysis, and an efficiency calculation for multiplex PCR optimization.J Clin Lab Anal. 2013; 27: 249-252Crossref PubMed Scopus (5) Google Scholar, 16Elnifro E.M. Ashshi A.M. Cooper R.J. Klapper P.E. Multiplex PCR: optimization and application in diagnostic virology.Clin Microbiol Rev. 2000; 13: 559-570Crossref PubMed Scopus (616) Google Scholar, 17Markoulatos P. Siafakas N. Moncany M. Multiplex polymerase chain reaction: a practical approach.J Clin Lab Anal. 2002; 16: 47-51Crossref PubMed Scopus (409) Google Scholar that are required to minimize primer dimers will not be required. Second, similar to the dual priming oligonucleotides mentioned earlier,14Chun J.Y. Kim K.J. Hwang I.T. Kim Y.J. Lee D.H. Lee I.K. Kim J.K. Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene.Nucleic Acids Res. 2007; 35: e40Crossref PubMed Scopus (163) Google Scholar cooperative primers bind to two separate sequences. This 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. Cooperative primers, with their inverted design and polyethylene glycol linkers, actually may function better, because of reduced steric hindrance, when the capture and primer binding sites are separated by tens to hundreds of nucleotides. Methods for preventing primer dimer formation in PCR also may apply to any of the numerous isothermal amplification strategies that are competing with PCR for use in the laboratory and the clinic.18Craw P. Balachandran W. Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review.Lab Chip. 2012; 12: 2469-2486Crossref PubMed Scopus (518) Google Scholar Unfortunately, primer design for the widely used loop-mediated isothermal amplification (LAMP) reaction is complicated enough to require custom software19Torres C. Vitalis E.A. Baker B.R. Gardner S.N. Torres M.W. Dzenitis J.M. LAVA: an open-source approach to designing LAMP (loop-mediated isothermal amplification) DNA signatures.BMC Bioinformatics. 2011; 12: 240Crossref PubMed Scopus (50) Google Scholar; combining this with the complexity of cooperative primers will take some effort but the benefits may be worth the added difficulty. This technology also may have utility for next-generation sequencing protocols that use a PCR step to prepare the library to be sequenced. Reactions that generate fewer primer dimers should require less clean-up. Several novel ideas were combined to develop cooperative primers and they will have uses beyond those presented. In a few years, standard PCR might be performed with a single oligonucleotide that combines the forward and reverse primers into one molecule. The resulting amplicons could have interesting melting properties that can be exploited for better detection or differentiation. Primers with two 3′ ends also may find utility in DNA origami applications20Michelotti N. Johnson-Buck A. Manzo A.J. Walter N.G. Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology.Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2012; 4: 139-152Crossref PubMed Scopus (32) Google Scholar and this may lead in turn to better tools for diagnostics (see Hartman et al21Hartman M.R. Yang D. Tran T.N. Lee K. Kahn J.S. Kiatwuthinon P. Yancey K.G. Trotsenko O. Minko S. Luo D. Thermostable branched DNA nanostructures as modular primers for polymerase chain reaction.Angew Chem Int Ed Engl. 2013; 52: 8699-8702Crossref PubMed Scopus (65) Google Scholar for a similar idea). The demand for deep multiplexing amplification techniques is only going to increase. Although strategies such as cooperative primers will facilitate such protocols, they also highlight the complexity of these reactions at the molecular level. Even as we reduce primer dimers, we need to better understand the sources of nonspecific amplification. The community would be well served by efforts to better characterize all of the products of a multiplex amplification. Next-generation sequencing can characterize, in depth, the specific and nonspecific amplicons generated in a multiplex PCR reaction without the biases introduced by cloning.9Brownie J. Shawcross S. Theaker J. Whitcombe D. Ferrie R. Newton C. Little S. The elimination of primer-dimer accumulation in PCR.Nucleic Acids Res. 1997; 25: 3235-3241Crossref PubMed Scopus (263) Google Scholar As the costs of next-generation sequencing continue to decrease it is possible that such analysis will become a standard part of the development and optimization of a multiplex PCR. Cooperative primers will not solve the other vexing problem of PCR—amplicon contamination. However, it can be easier to detect false-positive than false-negative results in clinical samples. As Satterfield3Satterfield B.C. Cooperative primers: a 2.5 million-fold improvement in the reduction of nonspecific amplification.J Mol Diagn. 2013; 16: 162-172Google Scholar notes, it is entirely possible that primer dimers have caused unrecognized sporadic false-negative results (especially when detecting rare pathogens). Cooperative primers will add a power tool to the PCR toolkit that should prevent this occurrence. Cooperative Primers: 2.5 Million–Fold Improvement in the Reduction of Nonspecific AmplificationThe Journal of Molecular DiagnosticsVol. 16Issue 2PreviewThe increasing need to multiplex nucleic acid reactions presses test designers to the limits of amplification specificity in PCR. Although more than a dozen hot starts have been developed for PCR to reduce primer-dimer formation, none can stop the propagation of primer-dimers once formed. Even a small number of primer-dimers can result in false-negatives and/or false-positives. Herein, we demonstrate a new class of primer technology that greatly reduces primer-dimer propagation, showing successful amplification of 60 template copies with no signal dampening in a background of 150,000,000 primer-dimers. Full-Text PDF Open Archive