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T E C H N I C A L T I P S BRUNO FREY AND BERNHARD SUPPMANN B o e h r i n g e r M a n n h e i m G m b H , D e p a r t m e n t o f M o l e c u l a r B i o l o g y, Nonnenwald, D-82372 Penzberg, Germany Demonstration of the Expand PCR System’s Greater Fidelity and Higher Yields with a lac I-based PCR Fidelity Assay TM lac I lac Iq lac Iq Cut with Cla I Ligate DNA Transform bacterial cells ➱ Grow cells on agar plate ➱ 8 Perform PCR ➱ lac Iq Count blue colonies and white colonies Calculate error rate Figure 1 Schematic of the lacI-based fidelity assay. Dra II 3632 3500 500 3000 lac Z' Bla Materials and Methods Construction of pUCIQ17 The truncated lacI gene of pUC19 was substituted by a functional copy of lacIq. A 178 bp Pvu II-Afl III fragment was replaced by a 1121 bp DNA fragment encoding lacIq (Figure 2). The α-complementing E. coli strain DH5α (2), once transformed with the resulting plasmid pUCIQ17 (3632 bp), produces white (LACI+) colonies on LB plates containing ampicillin (100 µg/ml) and X-Gal (0.004% w/v). q ➱ ➱ ➱ Thermostable polymerases possessing a 3'→5' exonuclease, or “proofreading,” activity are often used in polymerase chain reactions because they offer higher fidelity amplification than enzymes lacking this activity. Unfortunately, these proofreading enzymes also produce substantially lower amplification yields than polymerases without proofreading activity. On the other hand, “non-proofreading” DNA polymerases (e.g., Taq DNA polymerase, Tth DNA polymerase) lack the high fidelity required for applications in which accurate replication is placed at a premium. Coupled with a recent breakthrough in PCR enzyme technology (1), these facts have led to the development of the Boehringer Mannheim ExpandTM PCR System, optimized mixtures of Taq DNA polymerase and the proofreading enzyme Pwo DNA polymerase. We wanted to demonstrate that the Expand PCR System offers a better combination of high yields and high fidelity amplification than non-proofreading enzymes (e.g., Taq DNA polymerase) or proofreading enzymes. Therefore, we performed a PCR fidelity assay (Figure 1) based on the amplification, circularization, and transformation of the pUC19 derivative pUCIQ17, which contains a functional lacIq allele (1). PCR-derived mutations in lacI result in a de-repression of the expression of lacZα and subsequent formation of a functional β-galactosidase enzyme, which can be easily detected on X-Gal indicator plates. pUCIQ17 3632 lac Iq 1000 2500 ori 2000 Figure 2 1500 Plasmid map of pUCIQ17. Template preparation pUCIQ17 was linearized by digestion with Dra II. A typical PCR reaction contained 5 or 10 ng of linearized, gel-purified plasmid DNA. PCR primers Both PCR primers used have Cla I cleavage sites at their 5' ends: Oligonucleotide CLA33 (34-mer, 24 matches: 5'-AGCTTATCGATGGCACTTTTCGGGGAAATGTGCG-3'), primes “left.” Oligonucleotide CLA55 (36-mer, 26 matches: 5'-AGCTTATCGATAAGCGATGCCGGGAGCAGACAAGC-3') primes “right” to the Dra II site of pUCIQ17 (pUC19). The length of the resulting PCR product is 3,493 bp. Polymerase chain reactions The polymerase chain reactions were performed according to the conditions detailed in the manufacturers’ instructions. We used the following cycling conditions for all DNA polymerase preparations except the Expand PCR System: ■ Denaturation: 10 s at 94°C ■ Annealing: 30 s at 57°C ■ Extension: 4 min at 72°C for 18 cycles. The following cycling conditions were employed for the Expand PCR System: ■ Denaturation: 10 s at 92°C ■ Annealing: 30 s at 57°C ■ Extension: 4 min at 68°C for 18 cycles. Purification and circularization of the 3.5 kb PCR products After the PCR, the amplification products were PEG-precipitated (without previous phenol treatment) according to Barnes (3). After a restriction digest with Cla I, the DNA was purified by gel electrophoresis. BIOCHEMICA ■ NO.2 [1995] T E C H N I C A L Ligation reactions were carried out with the Boehringer Mannheim Rapid Ligation Kit; DNA concentrations per ligation reaction did not exceed 30 ng. lacI-based assay The resulting PCR-derived plasmids were transformed in E. coli DH5α as described by Hanahan (2), and plated on LB Amp100 X-Gal plates. After incubation overnight at 37 oC, blue and white colonies were counted. The error rate (f) per bp was calculated with a rearranged equation published by Keohavong and Thilly (4): f = -lnF / d x b bp; where F is the fraction of white colonies: F = white (LACI+)/total colony number; d is the number of DNA duplications: 2d = output DNA/input DNA; and b is the effective target size of the (1080 bp) lacI gene, which is 349 bp according to Provost et al. (5); there are 349 phenotypically identified (by color screening) single-base substitutions (nonsense and mis-sense) at 179 codons (approximately 50% of the coding region) within the lacI gene (5). Frameshift errors, which may occur at every position in the 1080 bp open reading frame of lacI, are not taken into account because little information is available for the specific DNA polymerases used in PCR systems (except for Taq DNA polymerase, which has a frameshift error rate of about 1/10th of the base substitution error rate determined in a one-pass assay [6,7]). Religation control Fifty nanograms of Dra II-linearized, gelpurified pUCIQ17 DNA was religated, and an aliquot of the ligation reaction was transformed into DH5α. After incubation overnight, only 1 of 3750 growing colonies (0.027%) showed a blue (LACI–) phenotype on LB Amp X-Gal plates; therefore, formation of concatameric ligation products (with subsequent intramolecular recombination in E. coli that eliminates an additional origin of replication) seems to be a very rare event. Restriction analysis of PCR-derived plasmids isolated from blue colonies also confirmed that the LACI– phenotype originates in PCRderived mutations of lacI, but not in deleterious recombination events after transformation of the ligated DNA in DH5α. BIOCHEMICA ■ NO.2 [1995] Results Table 1 illustrates that Boehringer Mannheim’s Expand PCR System mixtures offer better combinations of high amplification yield and high fidelity PCR. They feature lower error rates than the enzymes lacking proofreading activity, such as the Taq DNA polymerase preparations and Tth DNA polymerase. In addition, the Expand PCR System mixtures produce substantially higher yields than all proofreading DNA polymerases, even from smaller amounts of DNA template. All other commercially available polymerase mixtures tested thus far demonstrated error rates in the range of Taq DNA polymerase, except the variation has been between 2.9 x 10–5 and 2.1 x 10–5 (results not shown; publication in preparation). The lacI-based fidelity system described in this article represents a rapid, convenient method for evaluating error rates for DNA polymerases in PCR systems. Due to low background activity, it is possible to measure error rates in the range of 10–4 to 10–6. The calculated error rate for Taq DNA polymerase is in good agreement with the published value (8). ■ polymerase template amount [ng] 5 yield [ng] DNA duplications (d) 9.2 Product ExpandTM Long Template PCR System* (PCR 0.5–40 kb) ExpandTM High Fidelity PCR System*.† (PCR ≤5 kb) T I P S Cat. No. 1681 834 Size 100 U (30 reactions) 1681 842 500 U (150 reactions) 1732 641 100 U (30 reactions) 1732 650 500 U (150 reactions) See page 17 for local pricing. † Available after May 15, 1995. References 1. Barnes, W. M. (1994) PNAS USA 91:2216. 2. Hanahan, D. (1983) J. Mol. Biol. 166:557. 3. Barnes, W. M. (1992) Gene 112:29. 4. Keohavong, P. and Thilly, W. G. (1989) PNAS USA 86:9253. 5. Provost, G. S., Kretz, P. L., Hamner, R. T., Matthews, C. D., Rogers, B. J., Lundberg, K. S., Dycaico, M. J. and Short, J. M. (1993) Mut. Research 288:133. 6. Tindall, K. R. and Kunkel, T. A. (1988) Biochemistry 27:6008. 7. Eckert, K. A. and Kunkel, T. A. (1990) Nucleic Acids Research 18:3739. 8. Lundberg, K. S., Shoemaker, D. D., Adams, M. W. W., Short, J. M., Sorge, J. A. and Marthur, E. J. (1991) Gene 108:1. *Purchase of this product is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process for life science research in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. blue colonies LACI– 151 white colonies LACI+ 3066 total % LACI– number of colonies 3217 4.7 error rate (ER565) 1.5x10–5 ExpandTM Long 3000 Template PCR System1 ExpandTM High 5 3000 9.2 73 2695 2768 2.6 8.3x10–6 Fidelity PCR System1 Taq from BM3 5 1500 8.2 167 2202 2369 7.1 2.6x10–5 Taq from 5 1500 8.2 148 1917 2065 7.2 2.6x10–5 Supplier C3 Tth from BM3 5 2000 8.6 280 2970 3250 8.6 3.0x10–5 Pwo from BM2 10 300 5 22 3447 3469 0.6 3.2x10–6 commonly used 10 80 3 12 2245 2257 0.5 3.8x10–6 proofreading enzyme from Supplier A2 (native enzyme) commonly used 10 125 3.6 90 2527 2617 3.4 2.2x10–5 proofreading enzyme from Supplier B2 Table 1. Fidelity of different DNA polymerases in PCR. Enzyme preparations marked with a “1” are mixtures of two polymerases, one of which possesses proofreading activity and one of which lacks proofreading activity. Enzyme preparations marked with a “2” are single enzymes possessing proofreading activity. Enzyme preparations marked with a “3” are single enzymes lacking proofreading activity. 9