- Email: [email protected]
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 addition of monomers to the 30 end of the new strand. In contrast, standard chemical synthesis of nucleic acid occurs in the opposite direction, 30 to 50 , by the addition of nucleoside 30 -O-phosphoramidites to the 50 end of the oligonucleotide. However, nucleosides containing 50 -O- phosphoramidites are commercially available and can be used in conventional automated DNA synthesizers. Such reverse phosphoramidites extend the growing chain in the 50 to 30 direction.1 Reverse and standard phosphoramidites also have been combined in the same synthesis to make unusual DNA molecules with alternating 30 to 30 and 50 to 50 phosphodiester linkages.2 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 Satterfield3 in this issue of The Journal of Molecular Diagnostics. Satterfield3 used both standard and reverse phosphoramidites to synthesize novel oligonucleotides that contained two 30 ends, and no 50 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.
Primer Dimers The development of highly sensitive, highly multiplexed assays for detecting infectious agents4,5 or cancer genotyping6 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 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
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.7,8 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 al9 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 30 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.10 Physical methods have been used to prevent the reaction from starting before the components are brought to the annealing temperature.7,11 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 Satterfield3 and Lebedev et al12). These methods work to varying degrees but they all share one important defectdthey do not prevent the amplification of primer Accepted for publication January 9, 2014. Disclosures: M.A.P. and K.M.R. are employed by BioFire Diagnostics, Inc. Address correspondence to Mark A. Poritz, Ph.D., BioFire Diagnostics, Inc., 390 Wakara Way, Salt Lake City, UT 84108. E-mail: mark.poritz@ biofiredx.com
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) or 50 to 30 (B) through a polyethylene 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) indicates 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 synthesis steps depicted here.
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).
Cooperative Primers
Satterfield3 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 earlier9 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.
Satterfield’s3 attack on the primer dimer problem is based on novel primer designs that prevent the amplification of existing primer dimers. It builds on previous work13 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 oligonucleotides14; however, cooperative primers have a novel topologic feature that makes them uniquedthe capture oligonucleotide is downstream (30 ) 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 30 end) and the primer sequence in a headto-head configuration (50 to 50 , separated by polyethylene glycol linkers) (Figure 1A). This is achieved by synthesizing part of the DNA molecule using the aforementioned reverse phosphoramidites.
Satterfield3 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. Satterfield3 provides suggestions, based on thermodynamic arguments and on the initial PCR data, for what the respective
160
jmd.amjpathol.org
Future Steps
-
The Journal of Molecular Diagnostics
Preventing Primer Dimer Amplification 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)15e17 that are required to minimize primer dimers will not be required. Second, similar to the dual priming oligonucleotides mentioned earlier,14 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.18 Unfortunately, primer design for the widely used loop-mediated isothermal amplification (LAMP) reaction is complicated enough to require custom software19; 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.
Significance 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 30 ends also may find utility in DNA origami applications20 and this may lead in turn to better tools for diagnostics (see Hartman et al21 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
The Journal of Molecular Diagnostics
-
jmd.amjpathol.org
specific and nonspecific amplicons generated in a multiplex PCR reaction without the biases introduced by cloning.9 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 PCRdamplicon contamination. However, it can be easier to detect false-positive than false-negative results in clinical samples. As Satterfield3 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.
References 1. Claeboe CD, Gao R, Hecht SM: 30 -modified oligonucleotides by reverse DNA synthesis. Nucleic Acids Res 2003, 31:5685e5691 2. Koga M, Geyer SJ, Regan JB, Beaucage SL: The synthesis of alternating alpha,beta-oligodeoxyribonucleotides with alternating (30 –>30 )and (50 –>50 )-internucleotic linkages as potential therapeutic agents. Nucleic Acids Symp Ser 1993, 29:3e4 3. Satterfield BC: Cooperative primers: a 2.5 million-fold improvement in the reduction of nonspecific amplification. J Mol Diagn 2013, 16: 162e172 4. Mahony JB, Petrich A, Smieja M: Molecular diagnosis of respiratory virus infections. Crit Rev Clin Lab Sci 2011, 48:217e249 5. Navidad JF, Griswold DJ, Gradus MS, 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, 51:3018e3024 6. Beadling C, Neff TL, Heinrich MC, Rhodes K, Thornton M, Leamon J, Andersen M, Corless CL: Combining highly multiplexed PCR with semiconductor-based sequencing for rapid cancer genotyping. 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-copynumber amplifications. Nucleic Acids Res 1992, 20:1717e1723 8. Hsu JT, Das S, Mohapatra S: Polymerase chain reaction engineering. Biotechnol Bioeng 1997, 55:359e366 9. Brownie 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, 25:3235e3241 10. Alvarez-Fernandez R: Explanatory chapter: PCR primer design. Methods Enzymol 2013, 529:1e21 11. Poritz MA, Blaschke AJ, Byington CL, Meyers L, Nilsson K, Jones DE, Thatcher SA, Robbins T, Lingenfelter B, Amiott E, Herbener A, Daly J, Dobrowolski SF, Teng DH, Ririe KM: FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection. PLoS One 2011, 6:e26047 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 primers: a novel approach for improved PCR performance. Nucleic Acids Res 2008, 36:e131 13. Satterfield BC, West JA, Caplan MR: Tentacle probes: eliminating false positives without sacrificing sensitivity. Nucleic Acids Res 2007, 35:e76 14. Chun JY, Kim KJ, Hwang IT, Kim YJ, Lee DH, Lee IK, Kim JK: Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene. Nucleic Acids Res 2007, 35:e40
161
Poritz and Ririe 15. Camacho JL, Torres EM, Cadena C, Prieto J, Prieto LL, Torregroza DA: The use of factorial design, image analysis, and an efficiency calculation for multiplex PCR optimization. J Clin Lab Anal 2013, 27:249e252 16. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE: Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 2000, 13:559e570 17. Markoulatos P, Siafakas N, Moncany M: Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal 2002, 16:47e51 18. Craw P, Balachandran W: Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. Lab Chip 2012, 12:2469e2486
19. Torres C, Vitalis EA, Baker BR, Gardner SN, Torres MW, Dzenitis JM: LAVA: an open-source approach to designing LAMP (loop-mediated isothermal amplification) DNA signatures. BMC Bioinformatics 2011, 12:240 20. Michelotti N, Johnson-Buck A, Manzo AJ, Walter NG: Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2012, 4:139e152 21. Hartman MR, Yang D, Tran TN, Lee K, Kahn JS, Kiatwuthinon P, Yancey KG, Trotsenko O, Minko S, Luo D: Thermostable branched DNA nanostructures as modular primers for polymerase chain reaction. Angew Chem Int Ed Engl 2013, 52:8699e8702
162
jmd.amjpathol.org
-
The Journal of Molecular Diagnostics
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 addition of monomers to the 30 end of the new strand. In contrast, standard chemical synthesis of nucleic acid occurs in the opposite direction, 30 to 50 , by the addition of nucleoside 30 -O-phosphoramidites to the 50 end of the oligonucleotide. However, nucleosides containing 50 -O- phosphoramidites are commercially available and can be used in conventional automated DNA synthesizers. Such reverse phosphoramidites extend the growing chain in the 50 to 30 direction.1 Reverse and standard phosphoramidites also have been combined in the same synthesis to make unusual DNA molecules with alternating 30 to 30 and 50 to 50 phosphodiester linkages.2 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 Satterfield3 in this issue of The Journal of Molecular Diagnostics. Satterfield3 used both standard and reverse phosphoramidites to synthesize novel oligonucleotides that contained two 30 ends, and no 50 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.
Primer Dimers The development of highly sensitive, highly multiplexed assays for detecting infectious agents4,5 or cancer genotyping6 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 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
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.7,8 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 al9 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 30 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.10 Physical methods have been used to prevent the reaction from starting before the components are brought to the annealing temperature.7,11 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 Satterfield3 and Lebedev et al12). These methods work to varying degrees but they all share one important defectdthey do not prevent the amplification of primer Accepted for publication January 9, 2014. Disclosures: M.A.P. and K.M.R. are employed by BioFire Diagnostics, Inc. Address correspondence to Mark A. Poritz, Ph.D., BioFire Diagnostics, Inc., 390 Wakara Way, Salt Lake City, UT 84108. E-mail: mark.poritz@ biofiredx.com
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) or 50 to 30 (B) through a polyethylene 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) indicates 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 synthesis steps depicted here.
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).
Cooperative Primers
Satterfield3 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 earlier9 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.
Satterfield’s3 attack on the primer dimer problem is based on novel primer designs that prevent the amplification of existing primer dimers. It builds on previous work13 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 oligonucleotides14; however, cooperative primers have a novel topologic feature that makes them uniquedthe capture oligonucleotide is downstream (30 ) 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 30 end) and the primer sequence in a headto-head configuration (50 to 50 , separated by polyethylene glycol linkers) (Figure 1A). This is achieved by synthesizing part of the DNA molecule using the aforementioned reverse phosphoramidites.
Satterfield3 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. Satterfield3 provides suggestions, based on thermodynamic arguments and on the initial PCR data, for what the respective
160
jmd.amjpathol.org
Future Steps
-
The Journal of Molecular Diagnostics
Preventing Primer Dimer Amplification 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)15e17 that are required to minimize primer dimers will not be required. Second, similar to the dual priming oligonucleotides mentioned earlier,14 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.18 Unfortunately, primer design for the widely used loop-mediated isothermal amplification (LAMP) reaction is complicated enough to require custom software19; 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.
Significance 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 30 ends also may find utility in DNA origami applications20 and this may lead in turn to better tools for diagnostics (see Hartman et al21 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
The Journal of Molecular Diagnostics
-
jmd.amjpathol.org
specific and nonspecific amplicons generated in a multiplex PCR reaction without the biases introduced by cloning.9 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 PCRdamplicon contamination. However, it can be easier to detect false-positive than false-negative results in clinical samples. As Satterfield3 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.
References 1. Claeboe CD, Gao R, Hecht SM: 30 -modified oligonucleotides by reverse DNA synthesis. Nucleic Acids Res 2003, 31:5685e5691 2. Koga M, Geyer SJ, Regan JB, Beaucage SL: The synthesis of alternating alpha,beta-oligodeoxyribonucleotides with alternating (30 –>30 )and (50 –>50 )-internucleotic linkages as potential therapeutic agents. Nucleic Acids Symp Ser 1993, 29:3e4 3. Satterfield BC: Cooperative primers: a 2.5 million-fold improvement in the reduction of nonspecific amplification. J Mol Diagn 2013, 16: 162e172 4. Mahony JB, Petrich A, Smieja M: Molecular diagnosis of respiratory virus infections. Crit Rev Clin Lab Sci 2011, 48:217e249 5. Navidad JF, Griswold DJ, Gradus MS, 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, 51:3018e3024 6. Beadling C, Neff TL, Heinrich MC, Rhodes K, Thornton M, Leamon J, Andersen M, Corless CL: Combining highly multiplexed PCR with semiconductor-based sequencing for rapid cancer genotyping. 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-copynumber amplifications. Nucleic Acids Res 1992, 20:1717e1723 8. Hsu JT, Das S, Mohapatra S: Polymerase chain reaction engineering. Biotechnol Bioeng 1997, 55:359e366 9. Brownie 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, 25:3235e3241 10. Alvarez-Fernandez R: Explanatory chapter: PCR primer design. Methods Enzymol 2013, 529:1e21 11. Poritz MA, Blaschke AJ, Byington CL, Meyers L, Nilsson K, Jones DE, Thatcher SA, Robbins T, Lingenfelter B, Amiott E, Herbener A, Daly J, Dobrowolski SF, Teng DH, Ririe KM: FilmArray, an automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection. PLoS One 2011, 6:e26047 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 primers: a novel approach for improved PCR performance. Nucleic Acids Res 2008, 36:e131 13. Satterfield BC, West JA, Caplan MR: Tentacle probes: eliminating false positives without sacrificing sensitivity. Nucleic Acids Res 2007, 35:e76 14. Chun JY, Kim KJ, Hwang IT, Kim YJ, Lee DH, Lee IK, Kim JK: Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene. Nucleic Acids Res 2007, 35:e40
161
Poritz and Ririe 15. Camacho JL, Torres EM, Cadena C, Prieto J, Prieto LL, Torregroza DA: The use of factorial design, image analysis, and an efficiency calculation for multiplex PCR optimization. J Clin Lab Anal 2013, 27:249e252 16. Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE: Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 2000, 13:559e570 17. Markoulatos P, Siafakas N, Moncany M: Multiplex polymerase chain reaction: a practical approach. J Clin Lab Anal 2002, 16:47e51 18. Craw P, Balachandran W: Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. Lab Chip 2012, 12:2469e2486
19. Torres C, Vitalis EA, Baker BR, Gardner SN, Torres MW, Dzenitis JM: LAVA: an open-source approach to designing LAMP (loop-mediated isothermal amplification) DNA signatures. BMC Bioinformatics 2011, 12:240 20. Michelotti N, Johnson-Buck A, Manzo AJ, Walter NG: Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2012, 4:139e152 21. Hartman MR, Yang D, Tran TN, Lee K, Kahn JS, Kiatwuthinon P, Yancey KG, Trotsenko O, Minko S, Luo D: Thermostable branched DNA nanostructures as modular primers for polymerase chain reaction. Angew Chem Int Ed Engl 2013, 52:8699e8702
162
jmd.amjpathol.org
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The Journal of Molecular Diagnostics