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Cooperative Primers Publication The Journal of Molecular Diagnostics, Vol. 16, No. 2, March 2014 jmd.amjpathol.org See related Commentary on page 159. TECHNICAL ADVANCE Cooperative Primers 2.5 MillioneFold Improvement in the Reduction of Nonspecific Amplification Brent C. Satterfield From DNA Logix Inc, Bountiful, Utah Accepted for publication October 30, 2013. The increasing need to multiplex nucleic acid reactions presses test designers to the limits of amplifi- cation specificity in PCR. Although more than a dozen hot starts have been developed for PCR to reduce Address correspondence to Brent C. Satterfield, Ph.D., primer-dimer formation, none can stop the propagation of primer-dimers once formed. Even a small DNA Logix Inc, 585 W. 500 S., number of primer-dimers can result in false-negatives and/or false-positives. Herein, we demonstrate a Ste. 210, Bountiful, UT 84010. new class of primer technology that greatly reduces primer-dimer propagation, showing successful E-mail: [email protected]. amplification of 60 template copies with no signal dampening in a background of 150,000,000 primer- dimers. In contrast, normal primers, with or without a hot start, experienced signal dampening with as few as 60 primer-dimers and false-negatives with only 600 primer-dimers. This represents more than a 2.5 millionefold improvement in reduction of nonspecific amplification. We also show how a probe can be incorporated into the cooperative primer, with 2.5 times more signal than conventional fluorescent probes. (J Mol Diagn 2014, 16: 163e173; http://dx.doi.org/10.1016/j.jmoldx.2013.10.004) In the short time since its inception, PCR has become an after the initial denaturing step, allowing more primer-dimers to almost indispensable part of medical and diagnostic sci- be formed and propagated during amplification. Because the ences.1,2 In PCR, a short DNA segment, called a primer, number of possible primer-dimer products increases poly- anneals to a target nucleic acid segment and is extended by nomially with the number of primers in the reaction according the polymerase, amplifying the DNA present. Although to the function ðn2 þ nÞ=2wheren is the number of primers, rules have been described for the selection of primers,3 highly multiplexed tests can be difficult to design. With the primers often anneal to and amplify each other in an un- increasing demand for highly multiplexed assays to diagnose predictable manner, forming primer-dimers. This problem is cancers, drug resistance, and other disease, a solution must be compounded in multiplexed tests, where the large number found to the primer-dimer problem. of primers increases the probability of primer-dimer for- Herein, we report on cooperative primers, the first technol- mation exponentially.3 Primer-dimers compete with the ogy to prevent primer-dimer formation and propagation during target for the primers and can result in signal dampening, the actual amplification rounds (Figure 1). The primers consist false-negatives, or even false-positives if the nonspecific of short sequences that would ordinarily not amplify the tem- amplification product also interacts with a probe. plate. Because of the low primer melting temperature (Tm), To try to prevent formation of primer-dimers, more than a unless the capture sequence binds holding the primer in close dozen hot start methods have been invented, each of which proximity to the template, the primers will not amplify. The limits the activity of the polymerase before an initial denaturing 4e15 step. However, none of these can stop 100% of primer- Disclosure: B.C.S. is the owner of DNA Logix Inc., which owns the dimer formation. Furthermore, the hot start protection is lost rights to the cooperative primer technologies described herein. 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.2013.10.004 Satterfield Figure 1 Schematic showing cooperative primer mechanisms. The short, low-Tm primer does not amplify unless the capture sequence binds first. The polyethylene glycol linker connecting the primer and the capture sequence prevents the polymerase from extending all the way through the primer to the cap- ture sequence, thus retaining the short primer speci- ficity in every round of amplification. Nonspecific amplification products that do not contain sequences complementary to the capture sequence, such as primer-dimers, do not propagate. Because capture sequence hybridization is required during every round of amplification for the primer to bind, it results in an exponential reduction of nonspecificampliconsin contrast to traditional hot starts that are effective only before starting amplification. The polymerase extension of the cooperative primer linked to the 50 end of the capture sequence (A) favors displacement of the capture sequence, whereas extension of the cooperative primer linked to the 30 end of the capture sequence (B)favors50-30 exonuclease cleavage of the capture sequence. Cleavage of the capture sequence allows it to double as a sequence-specific probe, releasing a fluorophore on nuclease cleavage. Because the capture sequence must bind for the primer to extend, the efficiency of probe hybridization and the resultant cleavage are theoretically very high. polyethylene glycol linker connecting the primer and the cap- P. simium Howler in Saimiri, MRA-353G; P. knowlesi H ture sequence prevents the polymerase from extending through strain, MRA-456 G; and P. falciparum FCR-3/Gambia clone the capture sequence, retaining the primer specificity in each D-4, knobless, MRA-739G. round of amplification. Nonspecific amplicons that do not have a complementary region to the capture sequence, such as Oligonucleotide Design and Synthesis primer-dimers, are not propagated. Because this process is 16,17 repeated in every round of amplification, it results in an poly- Adapting math previously derived for cooperative probes nomial reduction of nonspecificamplification, in contrast to and neglecting entropic/enthalpic penalties due to restricting traditional hot starts, which are effective only before starting the mobility from the linker, the effective equilibrium constant reaction. In addition, when the capture sequence is labeled, it for cooperative primers can be expressed as follows: fi 0 can double as a sequence-speci c probe in reactions using a 5 - Ccap þ Cprimer þ Cboth 0 K ZK þ K þ P K K Z ð1Þ 3 exonuclease active polymerase. Because the capture eff primer cap L primer cap PT sequence must bind for the primer to extend, theoretically where K is the equilibrium constant, with the added sub- 100% of primer extensions also result in probe hybridization scripts cap, primer, both, and eff referring to capture and cleavage, increasing the signal-to-noise ratio. We hy- sequence, primer, both capture sequence and primer, and pothesized that these cooperative constructs would allow effi- effective, respectively. cient amplification of the template while simultaneously PZP À C À C À C ; TZT À C À C À C reducing the impact of primer-dimers on assay results. o cap primer both o cap primer both ð2Þ where P and T are initial primer and target concentrations, Materials and Methods o o respectively, PL refers to the local primer concentration Z Â Reagents [PL 1 molecule/(volume swept out by linker length Avogadro’s number)], and C is the concentration of the The following reagents were obtained through the Malaria sequence indicated by the subscript that is hybridized to the Research and Reference Reagent Resource Center as part of template. From this equation, the effective primer binding the Biodefense and Emerging Infections Research Resources efficiency (Eff ) in the initial rounds of amplification (eg, Po >> To) can be calculated as follows: Repository, National Institute of Allergy and Infectious Dis- À Á eases, NIH: Genomic DNA, P. falciparum St. Lucia, MRA- þ K þ P K K P ZCprimer CbothZ primer L primer cap 0 ð Þ 331G; P. vivax Panama in Aotus, MRA-343G; P. brasilianum Eff 3 T0 1 þ Keff P0 Peruvian III in Saimiri, MRA-349G; P. cynomolgi bastianellii in Rhesus, MRA-350G; P. cynomolgi Smithsonian in Rhesus, Exploration of predicted enthalpy and entropy values to MRA-351G; P. fragile-type strain in Rhesus, MRA-352G; evaluate the equilibrium constants in equation (1) reveals 164 jmd.amjpathol.org - The Journal of Molecular Diagnostics Cooperative Primers that primers with predicted Tms 7Cto12C below the performed by Biosearch Technologies (Petaluma, CA). Dual reaction temperature can still amplify with up to 99% effi- high-performance liquid chromatography purification was ciency when coupled with capture sequences with an equal used to purify the cooperative primers. Template was syn- or greater Tm and when separated by three or fewer hexa- thesized by either Biosearch Technologies or Integrated ethylene glycol sequences (HEGs). When six HEG linkers DNA Technologies (Coralville, IA) with no purification. separate the primer and the capture sequence, 99% effi- ciencies can still be obtained with predicted primer Tms 4 C Cooperative Primer PCR Efficiency to 7C below the reaction temperature. For proof of concept, cooperative primers were designed Human beta-actin real-time PCR was run by making a to the human beta-actin gene, the mitochondrial sequence of master mix with a 250-nmol/L final concentration of each Plasmodium spp., and the rpoB D516V mutation in Myco- primer/probe (b-act P, b-act cF, b-act cR), a 5-mmol/L final bacterium tuberculosis (Table 1). The primer sequence was concentration of MgCl2, and an additional 0.275 U per designed with a Tm 3Cto9C below the reaction tem- reaction of GoTaq polymerase in GoTaq colorless master perature. The capture sequence was designed with a Tm 3C mix (Promega Corp., Madison, WI). Dilutions of template to 11C below the reaction temperature or 5C and 7C were made using 600,000, 6000, 60, and 0 copies.
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