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Unwanted Mutations in PCR Mutagenesis: Avoiding the Predictable

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Unwanted Mutations in PCR Mutagenesis: Avoiding the Predictable Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Technical Tips PCR is a useful technique for generating merase terminal transferase-like activity Unwanted site-specific mutations (l'z) and recombi- generated mutations. Mutations in PCR nations (3~ that has distinct advantages Typical PCR mutagenesis and PCR re- over traditional techniques. Traditional combination protocols use first-round Mutagenesis: oligonucleotide-directed mutagenesis PCR products to prime a subsequent protocols create a mismatch at the de- round of PCR. These first-round PCR Avoiding the sired position between the template products may be modified at their 3' Predictable DNA strand and a complementary DNA ends. When used to prime a subsequent strand synthesized in vitro. When DNA round of PCR, the modified product containing the mismatch is transformed may substitute an unwanted deoxynu- into bacteria, the bacteria correct the cleotide adjacent to the 3' end of this Kelly D. Smith, 1'2 mismatch, either introducing the de- first-round product. Thus, Taq poly- sired mutation or restoring the original merase terminal transferase-like activity Alicia Valenzuela, 1 nucleotide. (4's) Because only a fraction may also cause unwanted mutations in Janet L. Vigna, ~'2 of the resulting colonies contain the mu- PCR. Here we document that, in contrast Kim Aalbers, ~ and tation, screening methods frequently are to misincorporations, Taq polymerase Charles T. Lutz ~ required to distinguish the desired mu- terminal transferase-like activity-gener- tations from the unmutated form. ated mutations are frequent. We also Departments of ~Pathology and PCR mutagenesis uses primers that present a mechanism to explain how 2Microbiology, University of Iowa, Iowa contain the desired mutations to amplify these mutations arise and how they can City, Iowa 52242 the template DNA. Therefore, all of the be avoided. amplified DNA contains the desired mu- tation on both strands, eliminating the requirement for a screening step. In ad- MATERIALS AND METHODS dition, PCR recombination uses overlap- ping primers to combine segments of Figure 1 shows the three methods of mu- DNA. This eliminates the requirement in tagenesis used. The first-round reaction traditional in vitro recombination meth- is essentially the same for all three meth- ods for restriction endonuclease sites at ods. Each 100-1~1 reaction mixture con- the point of recombination. tains 100 ng of plasmid DNA, 100 In PCR mutagenesis and recombina- pmoles of each oligonucleotide primer tion, the cloned products must be se- (synthesized on Applied Biosystems 391 quenced to identify any unwanted mu- PCR-Mate, deblocked, and purified on tations generated during PCR. Most Sephadex G-25 columns), 200 I~M dNTPs, researchers have reported a low fie- 50 mM KC1, 10 mM Tris-HC1 (pH 8.8 at quency of unwanted mutations, in the 25°C), 1.5 mM MgCl2, 0.1% Triton X-IO0, range of 0.026% °~ to 0.25% (6) per nucle- and 2.5 units of Taq polymerase otide. These unwanted mutations can be (Promega). Reactions are overlaid with attributed to Taq polymerase misincor- 100 t~1 of mineral oil and incubated in a porating deoxynucleotides during tem- thermocycler (MJ Research, Inc.) for 5 plate-dependent chain elongation. Thus, min at 94°C, then 30 sec at 50°C, 60 sec at Taq polymerase misincorporations are 72°C, and 60 sec at 94°C for 20 cycles, not frequent in PCR mutagenesis and re- followed by 30 sec at 50°C and 7 min at combination procedures. In addition to 72°C. polymerase activity, however, Taq poly- For two-step mutagenesis, separate merase has a terminal transferase-like ac- first-round reactions are performed for tivity. (7) This activity adds a deoxynucle- each of the two overlapping mutagenic otide to each 3' end of double-stranded primers. Twenty microliters of each first- DNA, including PCR products. Molecular round reaction mixture is separated on a techniques, such as DNA cloning and 1% low-melting-temperature agarose gel mutagenesis, that utilize PCR must com- (FisherBiotech) in TAE buffer, is) The spe- pensate for Taq polymerase terminal cific bands are excised and melted at transferase-like activity. To overcome 68°C immediately prior to use. In the problems in the direct cloning of PCR two-step procedure, the second round of products, new vectors (TA Cloning, In- PCR is identical to the first-round reac- vitrogen, and CloneAmp, GIBCO BRL) tion except that template DNA consists capitalize on Taq polymerase terminal of equimolar amounts of the agarose-iso- transferase-like activity. To overcome lated first-round amplification products problems in PCR mutagenesis and re- (5-50 ng of each in a total volume not combination, the investigator must un- more than 10 i~1) and the a and d primers derstand the mechanism of Taq poly- are used (Fig. 1). 2:253-257©1993 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/93 $3.00 PCR Methods and Applications 253 Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Technical Tipslllllll a. One Step FIGURE 1 PCR mutagenesis methods. (a) In RESULTS AND DISCUSSION a the one-step method, the mutagenic primer (d) contains both the desired substitution (tri- Unwanted nucleotide substitutions were angle) and a restriction endonuclease (R.E.) found in 38 (62%) of the 61 variants se- first round PCR site. An external primer (a) is located external quenced (single substitutions in 37 and to another R.E. site. After one round of PCR, two substitutions in 1) and fell into two the a + d product is digested and subcloned patterns. One pattern consists of appar- a+d product into a plasmid. (b) In the two-step method, ently random mutations distant from two complementary mutagenic primers (b R.E. digest the PCR primers. These occur infre- and c) and two external primers (a and d) are quently, in 3 out of 61 or 5% of the vari- used in two separate first-round reactions. In ants, and are most likely caused by Taq the second-round of PCR, a + b and c + d polymerase rnisincorporating deoxynu- products are combined with the external ligate to plasmid DNA cleotides during template-dependent primers, a and d. The resulting a + d product b. Two Step is digested and subcloned into a plasmid. (c) chain elongation. (1°'11> Misincorpora- In megaprimer mutagenesis, the mutagenic tion mutations were not detected in primer (b) and an external primer (a) are used products of the megaprimer method a L ~ d in the first-round of PCR to create the (4610 nucleotides sequenced from 26 "megaprimer", the a + b product. In the sec- clones) and occurred at a frequency of first round PCR ond round of PCR, the "megaprimer" and an 0.06% per nucleotide sequenced for each external primer (d) are used. The resulting of the one-step and the two-step proce- c+d product a + d product is digested and subcloned into dures, comparable to frequencies found a+b product [ a plasmid. In the figure, thick lines denote by others, 0.026%, (1> 0.06%, (2> and oligonucleotides and thin lines denote tem- L combine first roundJ 0.25%. (6> The fidelity of Taq polymerase plate or PCR synthesized DNA. under standard PCR conditions ranges from 0.1x 10 -4 to 2.1x 10 -4 errors/ a ~ - ~ • bp. (~-14> Therefore, the theoretical mu- For the megaprimer method, one tation frequency is predicted to be 0.04- second round PCR strand of the first-round PCR product 0.9% per nucleotide after 21 cycles of serves as a large primer for the second- amplification. Our frequency of errors falls within this theoretical range, and, a+d product round PCR. The first-round PCR mixture is concentrated by ethanol precipitation more importantly, the fidelity of Taq I R.E. digest and separated on a 1% LE agarose gel polymerase permits faithful replication (SeaKem) in TBE buffer(8~; the specific of DNA used in PCR mutagenesis. We band is excised, electroeluted, precipi- typically limit the number of essential ligate to plasmid DNA tated, and resuspended in H20. The sec- (i.e., amino acid coding) nucleotides to c. Megaprimer ond-round reaction for megaprimer mu- less than 300, thereby reducing the prob- ability of finding an error from Taq poly- a tagenesis is essentially the same as the first-round except 100-400 ng of the iso- merase misincorporation in any given lated first-round product, the "mega- clone. The three misincorporation mu- primer," is used in place of one of the tations detected are all transitions, two first round PCR oligonucleotide primers (Fig. 1). A:T-->G:C and one G:C--->A:T, consistent After cloning PCR products into plas- with previous observations that a+b product mids, mutations are analyzed by DNA se- A:T--~G:C mutations are the most com- combine first round product quencing using Sequenase kit version mon error by Taq polymerase. (11'~4> and template DNA 2.0 (United States Biochemicals) or cy- In contrast to misincorporations, the "megaprimer" cle-sequencing. (9> second group of mutations is common, A d TABLE 1 Taq Polymerase Terminal Transferase-like Activity Causes Most of the second round PCR Unwanted Mutations Seen in PCR Mutagenesis Unwanted mutations a+d product Number of mutants adjacent to distant from I R.E. digest sequenced Protocol Primers a Primersb Total 11 one step 0 1 1 ligate to plasmid DNA 24 two step 14 2 16 26 megaprimer 22 0 22 aConsistent with Taq polymerase terminal transferase-like activity causing the mutations listed in this column, according to the mechanism described in the text and in Fig."2.
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