Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Technical Tipslill|ll Pfu DNA polymerase is a novel thermo- of plasmid containing a rat GR cDNA as PCR with stable DNA polymerase that possesses a target sequence, 200 }J.MdNTPs, and 3 I~M Degenerate 3'--~ 5' exonuclease (proofreading) ac- of each primer. Taq DNA polymerase tivity responsible for a low error rate per buffer was 10 mM Tris-HCL (pH 8.3), 50 Primers nucleotide. Here, we show that degener- mM KCI, 2 mM MgC12, and 0.1 mg/ml gel- ate primers can also be used efficiently atin. Pfu DNA polymerase buffer was 9Containing with Pfu DNA polymerase for PCR. How- composed of 20 mM Tris-HCl (pH 8.8), Deoxylnoslne Fails ever, in contrast with Taq DNA poly- 10 mM KC1, 6 mM (NH4)2SO4, 2 mM merase, Pfu DNA polymerase cannot am- MgCI2, 0.1% Triton, and 0.1 mg/ml of with Pfu D NA plify a target sequence using degenerate BSA. Samples were amplified for 40 cy- primers substituted at a single position cles in a DNA thermocycler using the fol- Polymerase with deoxyinosine. lowing parameters: 94~ for 1 rain; 45~ PCR has become a powerful tool in for 2 min; and 72~ for 2 min. In the first molecular biology and is widely used for cycle incubation times were increased to enzymatic amplification of specific se- 5 min, and following the final cycle PCR Thomas Knittel and quences from small amounts of template was completed with a 13-min incubation Didier Picard DNA. Mixtures of primers representing at 72~ Four microliters of each reaction all codon choices of a target sequence was fractionated on a 2% agarose gel D~partement de Biologie Cellulaire, (degenerate primers) are often designed containing I iJ.g/ml of ethidium bromide Universit~ de Gen~ve, Sciences III, 1211 for PCR. (~ However, primer degeneracy and visualized by UV fluorescence. Gen~ve 4, Switzerland can reduce PCR efficiency and specificity dramatically. Therefore, substitution of degenerate primer positions with deoxy- RESULTS AND DISCUSSION inosine, which can base-pair with A, C, G, or T, has been used because it seems Using Taq or Pfu DNA polymerase and to increase PCR reliability and consis- degenerate primers A and B, PCR yielded tency.( ~2-4~ Recently, a new thermo- a single major DNA band of predicted stable DNA polymerase, termed Pfu DNA size (828 bp) (Fig. 1). A single deoxyinos- polymerase, has been isolated from the ine substitution in primer B abolished hyperthermophilic archaebacterium Py- amplification by Pfu DNA polymerase rococcus furiosus/s~ In contrast with Taq while Taq polymerase still worked with DNA polymerase, Pfu DNA polymerase comparable efficiency (Fig. 1). This strik- possesses a 3'---~ 5' exonuclease (proof- reading) activity that is responsible for >10-fold improvement in error rate per nucleotide/s~ At present, it remains to be clarified whether PCR works with Pfu DNA polymerase using degenerate olig- onucleotides or deoxyinosine-contain- ing primers. MATERIALS AND METHODS Degenerate sense (primer A) and an- tisense (primer B) oligonucleotide prim- ers corresponding to conserved amino acid sequences within the DNA and hor- mone-binding regions of the rat gluco- corticoid receptor (GR), respectively, had the following sequence: primer A, TG(C/ FIGURE 1 Pfu DNA polymerase fails to am- T) AA(G/A) GT(A/C/GfI') TT(CFF) TT(CfF) plify a target sequence using degenerate prim- AA; primer B, (T/C)TG (G/A)TA (G/A)AA ers with a single deoxyinosine substitution. (T/G/C/A)C(G/T) (T/C)TG CCA. Deoxyi- PCR reactions were performed using different nosine was incorporated at position 10 DNA polymerases and degenerate primers with (primer BI) or without (primers A and B) in antisense primer B, creating primer BI deoxyinosine substitution (see Materials and with the sequence (T/C)TG (G/A)TA (G/ Methods). Four microliters of each reaction A)AA IC(G/T) (T/C)TG CCA. PCR amplifi- was analyzed on a 2% agarose gel. (Lane 1) cation was carried out in a 20-1~1 reaction EcoRI/HindIlI-digested ~ DNA; (lanes 2,3) Taq mixture containing 0.5 units of Taq DNA DNA polymerase; (lanes 4,5) Pfu DNA poly- polymerase (Biofinex, Switzerland) or merase. (Lanes 2,4) Primers A + B; (lanes 3,5) Pfu DNA polymerase (Stratagene), 1 pg primers A + Bl (deoxyinosine substituted). $46 PCR Methods and Applications 2:346-3479 by ColdSpring Harbor Laboratory ISSN 1054-9803/93 $3.00 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press mWlllllll Technical Tips ing difference between the two poly- 3. Patil, R.V. and E.E. Dekker. 1990. PCR am- merases could not be overcome by vary- plification of an Escherichia coli gene us- ing PCR parameters (data not shown). ing mixed primers containing deoxyinos- Notably, we tested different primer sets ine at ambiguous positions in degenerate and annealing temperatures (40~ 45~ amino acid codons. Nucleic Acids Res. 18: 3080. 50~ different concentrations of prim- 4. Batzer, M.A., J.E. Carlton, and P.L. Deini- ers (0.3-6 p.M), template DNA (0.1-10 ger. 1991. Enhanced evolutionary PCR us- pg), dNTPs (100-200 p.M), magnesium ing oligonucleotides with inosine at the ions (1.5-6 raM), and Pfu DNA poly- 3' terminus. Nucleic Acids Res. 19: 5081. merase (0.25-1 units). The difference 5. Lundberg, K.S., D.D. Shoemaker, M.W.W. also held up with the PCR "hot start" Adams, J.M. Short, J.A. Sorge, and E.J. technique, ~6~ in the presence of 10% Mathur. 1991. High-fidelity amplification DMSO or when Pfu DNA polymerase was using a thermostable DNA polymerase added after the annealing of the primers isolated from Pyrococcus furiosus. Gene according to the manufacturer's advice. 108: 1-6. 6. Chou, Q., M. Russel, D.E. Birch, R. The fact that Pfu DNA polymerase Jonathan, and W. Block. 1992. Prevention cannot amplify a target sequence using of pre-PCR mis-priming and primer deoxyinosine-containing primers may dimerization improves low copy-number be the result of its 3' ~ 5' exonuclease amplifications. Nucleic Acids Res. activity. Pfu DNA polymerase might fail 20" 1717-1723. to incorporate a base complementary to deoxyinosine during consecutive rounds of PCR, preventing an exponential am- Received December I, 1992; accepted plification of target DNA. It is quite January 15, 1993. likely that other thermostable DNA poly- merases with proofreading activity would show the same limitations. How- ever, it should be emphasized that Pfu DNA polymerase did work under all con- ditions tested using degenerate primers without deoxyinosine substitution. Therefore, its lower error rate per nucle- otide, compared with that of Taq DNA polymerase, makes Pfu DNA polymerase a powerful tool for PCR employing de- generate primers, for example, in evolu- tionary PCR. ACKNOWLEDGMENTS We are grateful to Thomas Gerster (Basel, Switzerland) for suggesting the use of deoxyinosine-substituted primers. T.K. was supported by a grant from the Deutsche Forschungsgemeinschaft (Kni 328/1-1). This work was supported by the Swiss National Science Foundation and the Canton de Gen~ve. REFERENCES 1. Compton, T. 1990. Degenerate primers for DNA amplification. In PCR protocols: A guide to methods and applications (ed. M.A. Innis, D.H. Gelfand, J.J. Sninsky, and T.J. White), pp. 39-45. Academic Press, San Diego. 2. Knoth, K., S. Roberds, C. Poteet, and M. Tamkun. 1988. Highly degenerate, inos- ine containing primers specifically am- plify rare cDNA using polymerase chain reaction. Nucleic Acids Res. 16: 10932. PCR Methods and Applications 347 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press PCR with degenerate primers containing deoxyinosine fails with Pfu DNA polymerase. T Knittel and D Picard Genome Res. 1993 2: 346-347 Access the most recent version at doi:10.1101/gr.2.4.346 License Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the Service top right corner of the article or click here. To subscribe to Genome Research go to: https://genome.cshlp.org/subscriptions Copyright © Cold Spring Harbor Laboratory Press.