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PCR has revolutionized nucleic acid vapor barrier on the surface of the aque- Wax-embedded analysis in many scientific disciplines, ous mixture. With this approach, the PCR Reagents including molecular biology, medical di- wax must first be melted and solidified agnostics, population genetics, and fo- on top of the lower aqueous layer prior rensic analysis. (1~ PCR is a highly sensi- to addition of the missing compo- tive technique that can selectively nent(s). In a similar approach, wax is enrich for a specific target from a back- melted and solidified to cover the lower Patricia Blair, ground of nonrelated sequences. How- aqueous layer completely. (7~ The miss- Rama Ramanujam, and ever, if all of the reaction components ing reagent is then layered onto the wax Brent A. Burdick (nucleotides, buffer containing magne- cap, followed by two drops of mineral sium, primers, a thermostable DNA poly- oil. When the temperature of the tubes Pharmacia Biotech, Milwaukee, merase, and sample containing the tar- in the thermocycler exceeds the melting Wisconsin get to be amplified) are mixed at room temperature of the wax, the wax rises to temperature prior to thermocycling, pre- the top of the tube and the upper and mature mispriming events such as non- lower aqueous layers mix by convection. specific annealing of primers to nontar- Another variation of the hot start get nucleic acid sequences and the technique involves drying one of the re- production of primer oligomerization action components in trehalose and em- (primer dimer) artifacts may occur. (z~ In bedding the dried reactant in a wax these cases, the undesired nonspecific bead. (8~ The wax bead containing the products that are generated can interfere missing reactant (in this example, the with amplification and detection of the primers) is then added to the PCR reac- specific target DNA sequences, especially tion at room temperature, and the reac- when low-copy-number amplifications tant is released when the temperature in are performed. the thermocycler melts the wax. With Hot start is a recent variation of PCR this approach, there is no need to melt that discourages these pre-PCR misprim- the wax prior to assembling the reaction. ing events prior to initial denatur- Our approach to the problem of pre- ation. (3'4~ In hot start PCR, one of the PCR mispriming is simple and conve- essential reaction components is deliber- nient. We have cosolidified PCR re- ately excluded until the temperature of agents with wax, omitting either the the reaction exceeds the temperature at template or the DNA polymerase. With which the primers anneal to the target our approach, the reagents can be stored template. The result is typically en- conveniently at room temperature and hanced sensitivity and specificity in the delivered to a reaction by simply adding PCR reaction. (s~ In one version of hot water and the template or enzyme to the start PCR, the missing component is cosolidified reagents. Thermocycling is added to the tubes in the thermal cycler performed directly with no need to melt block at high temperature. Although this the cosolidified reagents prior to assem- method is simple, it involves manually bling the reaction. The reagents are re- opening the tubes once they have leased from the wax when the tempera- reached the appropriate temperature. ture reaches the melting point of the This is not only cumbersome and time- wax. All aqueous reagents mix, and the consuming when many samples are pro- wax then forms a vapor barrier on the cessed simultaneously but is also prone surface. We anticipate that this method to cross-contamination. will be useful in other amplification sys- Recent variations of hot start PCR in- tems with similar considerations. volve the use of paraffin wax to segregate We have expanded this approach to the reaction components in solution cosolidify nonthermostable reagents for and require step-wise assembly of the re- first-strand cDNA synthesis by reverse action mixture. All reagents are present transcriptase-PCR (RT-PCR). RT-PCR is in the tubes prior to thermocycling, a simple modification of PCR that allows thereby eliminating the need for open- for the selective amplification of ex- ing the tubes manually. One method in- tremely small amounts of target cludes an AmpliWax vapor barrier RNA. (9'1~ Cosolidification is particularly whereby a layer of solid wax is used to useful for preparing, storing, and deliv- separate the retained reagent(s) from the ering first-strand cDNA for eventual PCR bulk reaction mix until the initial dena- amplification when samples are avail- turation step melts the wax. (6) The upper able in isolated areas. Samples can be and lower aqueous layers are mixed by prepared easily, first-strand cDNA syn- convection, and the wax then forms a thesis performed using cosolidified re-

4:191-1949 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/94 $5.00 PCR Methods andAppllcations 191 Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Technical

agents, and the first-strand cDNA prod- stored at either room temperature or on 1 hr. For two samples, total RNA was uct cosolidified in the wax for ice for 2 hrs. For each sample, the miss- added to a first-strand reaction and tubes convenient storage when PCR amplifica- ing component and 93 i~l of sterile dis- were incubated at 65~ for -2 rain prior tion is not performed immediately. This tilled water were added to the other re- to the 1 hr incubation at 37~ For the method also allows for simple delivery agents in solution or to the cosolidified two tubes containing cosolidified first- of the stored product to a subsequent wax preparation. An AmpliWax PCR strand reagents, 20 I~l of total RNA was PCR reaction. Gem 100 was added to those tubes that added to each tube and the tubes were did not contain cosolidified reagents. heated at 65~ for I min to melt the wax. Tubes were placed into a thermocycler The tubes were then vortexed to mix the MATERIALS AND METHODS (Perkin-Elmer, DNA Thermal Cycler sample with the reagents and incubated Preparation of Cosolidifled 480), and PCR was performed using the at 65~ for an additional minute to Reagents following program: 30 cycles of 94~ for remelt the wax. The tubes were then 1 min, 55~ for 2 min, 72~ for 2 min. placed at 37~ for 1 hr. The wax formed Cosolidified PCR reagents were prepared a solid layer over the top of the aqueous as follows: For each sample, selected PCR reactants. Fifty pmoles of each primer Preparation of Whole Blood for reagents (in a volume of 6 or 7 i~l) were specific for human [3-globin (PCO4 and First-strand cDNA Synthesis mixed in a 0.5 ml Eppendorf tube just GH20), 64 i~l of sterile distilled water, prior to cosolidification. A single Ampli- Whole human blood (EDTA-treated) was and 1 I~l (5 units) of AmpliTaq were Wax PCR Gem 100 (Perkin-Elmer Cetus) prepared for first-strand cDNA synthesis added to each sample. An AmpliWax was melted in a separate 0.5 ml micro- as described by Shi and Liu. <11> For each PCR Gem 100 was added to the samples centrifuge tube by incubating the tube at of five samples, 90 ~l of RNase-free water that did not contain cosolidified re- 65~ for -2 min. The mixed PCR re- (treated with diethylpyrocarbonate) was agents. Tubes were placed into a thermo- agents were then added to the side of the added to 10 i~l of whole human blood in cycler (Perkin-Elmer, DNA Thermal Cy- tube containing the melted wax, and the a 0.5 ml microcentrifuge tube. Samples cler 480), and PCR was performed using tube was tapped until the reagents were were incubated on ice for 5 min to lyse the following program: 30 cycles of 94~ in contact with the wax. The tube was the erythrocytes and centrifuged at 6000 for 1 min, 55~ for 2 min, and 72~ for 2 then placed at 65~ just until the wax rpm in a microcentrifuge for 10 min. min. melted (-1 min). The PCR reagents and The supernatants were discarded. Each wax cosolidified following vortexing. Al- pellet was resuspended in 20 ~l of RNase- RESULTS AND DISCUSSION ternatively, PCR reagents could be pre- free water containing 24 units of RNA- dispensed into tubes containing solid guard ribonuclease inhibitor (Pharma- To demonstrate that cosolidified PCR re- wax and then cosolidified by melting cia). Samples were incubated on ice for agents are useful for decreasing nonspe- and vortexing. 20 rain to release total RNA from the leu- cific, higher-molecular weight products Cosolidified first-strand cDNA syn- kocytes. Immediately prior to first- in PCR reactions, all PCR reagents, ex- thesis reagents were prepared as de- strand cDNA synthesis, samples were in- cept enzyme, were either cosolidified scribed above, except 11 i~l of first-strand cubated at 65~ for 10 min to denature with wax or left in solution and stored at cDNA synthesis reagents containing oli- the template RNA. room temperature or on ice. For each go(dT)12-18 primer (cDNA Synthesis sample, 5 units of AmpliTaq and 93 i~l of Kit, Pharmacia) were cosolidified with sterile distilled water were added to the RT-PCR wax. other reagents in solution or on top of The composition of the first-strand mix- the cosolidified wax preparation. Nega- ture is as follows (from cDNA synthesis tive (containing no template) and posi- PCR Amplification kit, Pharmacia): 136 mM Tris-HC1 (pH tive control reactions were also prepared The following reagents were used for 8.3), 204 mM KCl, 27 mM MgC12, 27 mM using reagents stored at -20~ Tubes PCR amplification in the experiments in- DTT, 0.2 mg/ml of BSA, 5.4 mM each of were placed into a thermocycler, and volving cosolidified PCR reagents: dATP, dCTP, dGTP, and dTTP, 60 units of PCR amplification was performed as de- pBR322, linearized with PvuII (1 ng/i~l), 1 Moloney murine leukemia virus (M- scribed. Figure 1 shows the results of this I~l; SF-4 primer, 5'-GATAAGCTTFAAT- MuLV) reverse transcriptase (cloned), 30 experiment. Using this model system, a GCGGTAGTITATCACAG-3', (50 pmoles/ units of RNAguard, and 5 t~g of oli- specific product of -350 bp is expected. w/l, 1 i~l; SF-5 primer, 5'-AGAGGATCCA- go(dT) 12-18. No visible PCR product is present in the CAGGACGGGTGTGGTCGCCA-3', (50 The following reagents were used for negative control lane (lane 1), indicating pmoles/l~l), 1 I~l; 50x buffer, 500 mM amplification of first-strand cDNA: PCO4 that cross-contamination of samples was Tris-HC1 (pH 8.3), 2.5 M KC1, 75 mM primer (Perkin-Elmer), 5'-CAACTTCATC- not a problem. All other lanes show a MgCl2, 2 I~l; DNA polymerization mix CACGTTCACC-3'; GH20 primer (Perkin- specific PCR product with the expected (Pharmacia), 20 mM each of dATP, dCTP, Elmer), 5'-GAAGAGCCAAGGACAGGTA- size and intensity equivalent to that of dGTP, and dTTP, 1 i~l; and AmpliTaq C-3'; AmpliTaq DNA polymerase. the positive control lane (lane 2). DNA polymerase (5 U/l~l), 1 ~l. For each sample, the entire volume Samples in which PCR reagents were Prior to PCR amplification, all PCR re- (20 i~l) of total RNA was used in a first- stored in solution, however (lanes 9-14), agents, except enzyme or template, were strand cDNA synthesis reaction. For one also indicate the presence of undesir- either cosolidified in wax or left in solu- sample, total RNA was added to a first- able higher-molecular-weight products. tion. Triplicate samples of each were strand reaction and incubated at 37~ for These nonspecific products are absent in

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samples prepared using reagents cosolid- ifed with wax (lanes 3-8). If all PCR components except the template are cosolidified, specificity of the reaction can be enhanced. An experiment similar to the one described above was performed, except the template, rather than the enzyme, was excluded. Triplicate samples were incubated at room temperature or on ice for 2 hr. Prior to PCR amplification, 1 ng of pBR322-PvuII and 93 i~l of sterile distilled water were added to the other reagents. Thermocycling was as described. It is evident from the results shown in Figure 2 that nonspecific, lower-molecular-weight products (primer-dimers) were generated when reactants were stored in solution (lanes 7-12). This problem was avoided, however, by using reactants cosolidified with wax (lanes 1-6). Also, the intensity of the specific FIGURE 1 PCR amplification using reagents (excluding enzyme) in wax or in solution, l~eactlons PCR product is somewhat reduced in were prepared in triplicate (except for negative and positive control). Ten rnicroliters of each PCR those lanes where primer-dimers are reaction was electrophoresed on a 1% agarose gel containing ethidium bromide to visualize the present, suggesting that formation of ar- -350-bp fragment. (Lane 1) Fresh reagents excluding template (negative control); (lane 2) fresh reagents including template (positive control); (lanes 3-5) reagents in wax, stored on ice; (lanes tifacts may deplete the reaction. 6-8) reagents in wax, stored at room temperature; (lanes 9-11) reagents in solution, stored on ice; In both of the experiments described (lanes 12-14) reagents in solution, stored at room temperature; (lane 15) 100 Base-Pair Ladder above using a model system, cosolidifi- (Pharmacia). cation of PCR reagents appeared to sig- nificantly reduce or eliminate nonspecific products that arise from premature interaction of reagents. From these data, it is reasonable to assume that if reagents are cosolidified with wax, their diffusion is greatly impeded and thus the proba- bility of their interaction is reduced. To show that cosolidification of nonthermostable reactants with wax is also feasible, first-strand cDNA reagents were cosolidified with wax as described in Materials and Methods. Five samples of total RNA were prepared from whole human blood and RT-PCR was performed as described. Figure 3, lanes 4 and 5, shows a specific PCR product (268 bp) equivalent in intensity to the control reaction where first-strand reagents were not subjected to an initial incubation at 65~ (lane 1). The reagents comprising the first-strand mixture can thus tolerate brief incubation at 65~ a prerequisite to cosolidification with wax. Lanes 2 and 3 show that a significant amount of specific PCR product can be generated from reactions performed with cosolidified first-strand reagents, despite the fact that FIGURE 2 PCR amplification using reagents (excluding template) in wax or in solution. Reactions were prepared in triplicate. Ten microliters of each PCR reaction was electrophoresed on a 1% the reaction components may be consid- agarose gel containing ethidium bromide to visualize the -350-bp fragment. (Lanes 1-3) Re- ered thermolabile. agents in wax, stored on ice; (lanes 4-6) reagents in wax, stored at room temperature; (lanes 7-9) In summary, we have developed an reagents in solution, stored on ice; (lanes 10-12) reagents in solution, stored at room tempera- extremely simple and convenient ture; (lane 13) 100 Base-Pair Ladder. method for preparing, storing, and deliv-

PCR Methods and Applications 193 Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Technical

from the cosolidified mixture, and sev- 9. Veres, G., R.A. Gibbs, S.E. Scherer, and eral preparations were more stable upon C.T. Caskey. 1987. The molecular basis of storage than similarly stored samples in the sparse fur mouse mutation. Science solution (data not shown). 237: 415-417. We have also successfully demon- 10. Kawasaki, E. 1990. Amplification of RNA. In PCR Protocols: A guide to methods and strated the principle that cosolidification applications (ed. M.A. Innis, D.H. Gelfand, can be used for more complex mixtures J.J. Sninsky, and T. White), pp. 21-27. Ac- such as first-strand reaction mixes. The ademic Press, San Diego, CA. use of such cosolidified reagents alone or 11. Shi, Y-J. and J-Z Liu. 1992. Direct reverse in combination with reagents dried in transcription-polymerase chain reaction the presence of a carbohydrate poly- from whole blood without RNA extrac- mer (12) should be amenable to automa- tion. GATA 9: 149-150. tion and should facilitate the application 12. Ramanujam, R., J. Heaster, C. Huang, J. of all aspects of PCR in routine practice. Jolly, J. Koelbl, C. Lively, E. Ogutu, E. Ting, S. Treml, B. Aldous, R. Hatley, S. Mathias, F. Franks, and B. Burdick. 1993. ACKNOWLEDGMENTS Ambient-temperature-stable molecular biology reagents. BioTechniques 14: 470- We thank David Boyer for valuable tech- 475. nical assistance and Dr. Glenda Mer- naugh for her critical review of the manuscript. Received June 27, 1994; accepted in revised form September 6, 1994.

REFERENCES 1. Saiki, R.K., S. Scharf, F. Faloona, K.B. Mul- FIGURE 3 RT-PCR using first-strand reagents lis, G.T. Horn, H.A. Erlich, and N. Arn- in wax or in solution. RNA isolated from 10 ~l heim. 1985. Enzymatic amplification of of whole human blood was used as a template ~-globin genomic sequences and restric- for first-strand cDNA synthesis. PCR amplifi- tion site analysis for diagnosis of sickle cation was performed following first-strand cell anemia. Science 230: 1350-1354. synthesis. Primers specific for human 2. Li, H., X. Cui, and N. Arnheim. 1990. Di- ~-globin were used for amplification. Ten mi- rect electrophoretic detection of the al- croliters of each PCR reaction was electro- lelic state of single DNA molecules in hu- phoresed on a 1% agarose gel containing man sperm by using the polymerase ethidium bromide to visualize the 268-bp chain reaction. Proc. Natl. Acad. Sci. fragment. (Lane 1) Fresh first-strand mix; 87: 4580-4584. (lanes 2-3) first-strand mix in wax; (lanes 4-5) 3. Erlich, H.A., D. Gelfand, and J.J. Sninsky. first-strand mix in solution, heated for 2 min- 1991. Recent advances in the polymerase utes at 65~ (lane 6) 100 Base-Pair Ladder. chain reaction. Science 252: 1643-1651. 4. Mullis, K.B. 1991. The polymerase chain reaction in an anemic mode: How to avoid cold oligodeoxyribonuclearfusion. ering PCR reagents. Using a model sys- PCR Methods Applic. 1: 1-4. tem, the utilization of reaction compo- 5. D'Aquila, R.T., L.J. Bechtel, J.A. Videler, nents cosolidified with wax resulted in J.J. Eron, P. Gorczyca, and J.C. Kaplan. significant reduction of nonspecific 1991. Maximizing sensitivity and specificity of PCR by pre-amplification heating. products, of both high and low molecu- Nucleic Acids Res. 19: 3749. lar weight, that can interfere with the 6. Chou, Q., M. Russell, D.E. Birch, J. Ray- generation and/or detection of specific mond, and W. Bloch. 1992. Prevention of PCR products. Samples can be prepared pre-PCR mis-priming and primer dimer- in minutes with no special equipment ization improves low-copy-number am- required. Delivery of reagents to a PCR plifications. Nucleic Acids Res. 20: 1717- reaction is direct with no requirement 1723. for the melting of the wax prior to PCR 7. Bassam, B.J. and G. Caetano-Anolles. amplification. 1993. Automated "Hot Start" PCR using We have extended this method of co- mineral oil and paraffin wax. BioTech- niques 14: 31-33. solidification to other molecular biology 8. Kaijalainen, S., P.J. Karhunen, K. Lalu, reagents, including restriction enzymes, and K. Lindstrom. 1993. An alternative DNA polymerases, modifying enzymes, hot start technique for PCR in small vol- and enzyme-conjugated antibodies (data umes using beads of wax-embedded reac- not shown). These reagents exhibited tion components dried in trehalose. Nu- significant activity following delivery cleic Acids Res. 21: 2959-2960.

194 PCR Methods and Applications Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press

Wax-embedded PCR reagents.

P Blair, R Ramanujam and B A Burdick

Genome Res. 1994 4: 191-194

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