Downloaded from genome.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Technical Presence of residual reverse transcriptase molecules, suggesting that enzymatic ex- Reverse (RT) activity in cDNA used for PCR re- tension of primer molecules may be re- Transcriptase Can sults in accumulation of primer-dimers sponsible for their increased tendency to and in inhibition of amplification of a form dimers and inhibition of amplifica- Inhibit PCR and specific DNA product. Inactivation of RT tion of specific DNA products. by heat or some other method solves Stimulate both these problems. We suggest that RT Primer-dimer is capable of adding nucleotides at the 3' MATERIALS AND METHODS end of primers before PCR amplification cDNA of Sabin 3 poliovirus was synthe- Formation begins, thereby extending short comple- sized by Moloney murine leukemia virus mentary regions in primers facilitating (M-MuLV) RT (GIBCO-BRL) with ran- their interaction. RT should be inacti- dom hexanucleotide primers on a tem- vated in all protocols involving PCR am- plate of RNA extracted from the virus. Konstantin M. Chumakov plification of cDNA. Conditions of this reaction were de- PCR amplification of cDNA synthe- scribed previously in detail. (2'3) Two seg- Center for Biologics Evaluation and sized in vitro by reverse transcriptase ments of type 30PV cDNA spanning nu- Research, Food and Drug (RT-PCR) is used widely in studies of cleotides 431-513 (segment 1) and Administration, Bethesda, both cellular and viral RNAs, enabling 2463-2603 (segment 2) were PCR ampli- Maryland 20852-1448 the powerful tools of DNA research to be fied in this study using primers listed in used for studies of RNA.(1~ One of the Table 1. In both cases, sense primers applications of RT-PCR is to detect and were taken in a lO-fold excess over an- quantitate neurovirulent revertants in tisense primers (final concentrations of oral poliovirus vaccine (OPV).(2'3~ Mu- 3 i~g/ml and 0.3 i~g/ml, respectively) to tant analysis by PCR and restriction en- ensure accumulation of predominantly zyme cleavage (MAPREC), developed in single-stranded DNA. (4~ Forty PCR cycles our laboratory for this purpose, involves were done (15 sec at 94~ 15 sec at 55~ synthesis of poliovirus cDNA by RT, and and 1 min at 72~ The second strand amplification of the DNA segment under was synthesized by adding 32p-labeled study by PCR with simultaneous cre- antisense primer and extending it with ation of restriction sites that are affected Taq DNA polymerase at 72~ Radioac- by the mutation of interest. While using tively labeled DNA was analyzed in 7.5% this method for quantitation of numer- polyacrylamide gel electrophoresis (PAGE). ous mutations in three types of OPV, we found that some crude cDNA prepara- tions (i.e., products of RT reactions with- RESULTS AND DISCUSSION out any further treatment) contained Inhibition of PCR inhibitor(s) that blocked PCR amplifica- tion of DNA. Dilution of such cDNA If the PCR reaction is primed with a samples restored PCR amplification. An- freshly made cDNA, the amplification is other problem with crude cDNA samples often poor, as evidenced by both the ab- was the apparent enhancement of" the sence or lower quantities of DNA prod- formation of artifactual DNA products, uct, and by large amounts of residual un- presumably primer-dimers. In this short used primers. A 5- to lO-fold dilution of communication, we report that both of cDNA prior to its addition to PCR re- these problems are caused by RT itself stored the amplification. Phenol extrac- and not by any other component of the tion/alcohol precipitation of cDNA also RT reaction mixture or the RT storage yielded cDNA that did not inhibit ampli- buffer. Phenol extraction or heating of fication. To discover whether this phe- newly synthesized cDNA solved both of nomenon is caused by RT or some com- these problems. Incubation of primers ponent present in cDNA preparation, with RT and dNTPs resulted in the incor- PCR mixtures were seeded with a DNA poration of [o~-32p]dATP into primer segment spanning nucleotides 431-513 TABLE 1 Primers Used in This Study pS-3/470.1 431 TGAGCTACAT GAGAGTGCTC CGGCCCCTGA ATGCGGCTGA 470 pA-3/484 513 CAGGCTGGCT GCTGGGTTGC AGCTGCCTGC 484 pS-3/2491 2463 CTGCGCTTCC ACAGGGTATT GAAGAATCG 2491 pA-3/2574 2603 TGAGTGCAGG TACCTCCTTG GAATGCGCCG 2574 62 PCR Methods and Applications 4:62-649 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/94$5.00 Downloaded from genome.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Technical Tips former one. To explain these effects of RT, it was suggested that it adds nucle- otides to the 3' end of primers, changing their sequence and specificity. Appar- ently, this occurred within a few minutes after all of the PCR components were combined and before amplification be- gan, as the first step in PCR is heating to 94~ To determine whether RT can add nucleotides to primers, primers used for amplification of segment 2 were incu- bated with RT and [~-3zP]dATP at room temperature. Analysis of reaction prod- ucts in PAGE clearly demonstrated that dATP was incorporated into primers and that this incorporation was dependent FIGURE 1 Effect of RT on PCR amplification of DNA. (A) PCR was seeded with DNA segment on the presence of active RT (results not corresponding to nucleotides 431-513 of type 30PV (segment 1), and RT was added at the shown). indicated concentrations. (B) Heat-inactivated RT (5 min at 94~ was added to samples. Control Inhibition of PCR and stimulation of is a sample with no template DNA or RT added. 32p-Labeled PCR product was separated from the primer-dimer formation described in primers by PAGE. this communication are likely to result from enzymatic modification of primers by RT. At room temperature, primers can of type 3 poliovirus genome (segment 1), DNA segment 2 (nucleotides 2463-2603) form unstable short-lived complexes with and decreasing amounts of RT were of type 3 poliovirus gene, another type each other by the annealing of short added to a row of eight tubes. Figure 1A of problem caused by cDNA was ob- complementary nucleotide stretches. Be- shows that no amplification occurred in served. In this case, primer-dimer was cause RT readily uses DNA as a tem- the first two tubes containing 15 and 3 the predominant PCR product in a sam- plate (s) and is known to use very short U/~I of RT. The inhibition was caused by ple to which no DNA template had been oligonucleotide primers to initiate DNA RT and not by components of the en- added. Figure 2 shows that adding as lit- strand synthesis, it is possible that it can zyme storage buffer, because RT heated tle as 0.001 U/~I of RT strongly stimu- add nucleotides to the 3' end of primers for 5 min at 94~ had no inhibitory ef- lated primer-dimer formation. Adding in these unstable complexes. Hence, it fect (Fig. 1B). Heat inactivation of cDNA RT heated to 94~ had no effect. may extend complementary stretches samples that otherwise inhibited PCR The above results demonstrate that and facilitate further primer-dimer for- also restored their activity. Therefore, the presence of active RT in a PCR mix- mation during PCR. The same mecha- the active RT rather than any compo- ture can inhibit specific amplification of nism may lead to inhibition of PCR if a nents of the storage buffer inhibits PCR. DNA and stimulate accumulation of substantial fraction of primers becomes primer-dimers. It should be noted that modified and therefore fails to bind the Enhancement of the latter process occurs only with se- template at specific sites. The latter effect Prlmer-dimer Formation lected primer pairs that are prone to should require much higher activity of dimer formation and that it requires RT. It is not known whether the PCR in- In an experiment with amplification of much lower amounts of RT than the hibition and stimulation of primer- dimer formation can be caused by RT of any source or whether these effects are specific to the Moloney RT that I have used. The practical implication of these findings is clear. When cDNA is used to prime PCR, RT should be inactivated ei- ther by phenol extraction or heating. ACKNOWLEDGMENTS This work was supported in part by grant 9102A01 from the National Vaccine Pro- gram Office. FIGURE 2 Stimulation of primer-dimer formation by RT. Varying amounts of RT were added to PCR containing no template DNA. Primers used for amplification of DNA segment 2 (nucleotides REFERENCES 2463-2603 of type 30PV) were used in this reaction. (Control) A sample with no template DNA 1. Kawasaki, E.S. 1990. Amplification of or RT added. 32P-Labeled PCR product was analyzed by PAGE. RNA. In PCR protocols: A guide to methods PCR Methods and Applications 63 Downloaded from genome.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Technical Tipslllllll and applications. (ed. M.A. Innis, D.H. Gel- land, J.J. Sninsky, and T.J. White), pp. 21- 27. Academic Press, San Diego, CA. 2. Chumakov, K.M., L.B. Powers, K.E. Noonan, I.B, Roninson, and I.S. Leven- book. 1991. Correlation between amount of virus with altered nucleotide sequence and the monkey test for acceptability of oral poliovirus vaccine. Proc. Natl. Acad. Sci. 88: 199-203. 3. Chumakov, K.M., L.P. Norwood, M.L. Parker, E.M. Dragunsky, Y. Ran, and I.S. Levenbook. 1992. RNA sequence variants in live poliovirus vaccine and their rela- tion to neurovirulence.