Mutagenic Oligonucleotide-Directed PCR Amplification (Mod-PCR): an Efficient Method for Generating Random Base Substitution Mutations in a DNA Sequence Element

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Mutagenic Oligonucleotide-directed PCR Amplification (Mod-PCR): An Efficient Method for Generating Random Base Substitution Mutations in a DNA Sequence Element

Lillian W. Chiang, 1'2 lulia Kovari, 1 and Martha M. Howe ~

1Department of Microbiology and Immunology, The University of Tennessee--Memphis, Memphis, Tennessee 38163; 2Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706

Saturation mutagenesis is one ap- mutations in the important regions thesis of long oligonucleotides to span proach for determining the contribu- require further characterization by the region between preexisting restric- tions of individual base pairs to the DNA sequence analysis. tion sites. (3-4) structure and function of defined Mutagenesis during PCR occurs natu- DNA sequence elements. In this pa- rally at a low frequency of -0.2% per per, we describe a novel method for nucleotide ~5) and can be increased to saturation mutagenesis involving A complete dissection of a DNA se- -2% under suboptimal reaction condi- PCR amplification with degenerate quence element, such as the binding site tions~6); therefore, it is inefficient for the synthetic oligonucleotides as prim- of a protein previously defined by foot- saturation mutagenesis of a small 10-bp ers. The degeneracy is confined to a printing, includes the isolation of every element. specific target within the primer by possible single point mutation within In this paper, we describe a mutagen- mixing a low percentage of the three the sequence of interest and the analysis esis procedure that involves PCR ampli- non-wild type (non-WT) nucleotide of the resulting phenotypic effects. Cur- fication with degenerate oligonucleotide precursors with WT at specific posi- rent methods for generating such muta- primers. This method reduces the cost of tions during primer synthesis. PCR tions include site-directed mutagenesis saturation mutagenesis by reducing the amplification of WT template DNA by specific synthetic oligonucleotides, ~) length of the oligonucleotides synthe- with the degenerate primer and an random mutagenesis with degenerate sized. It also allows for efficient mutant opposing WT primer, followed by oligonucleotides, ~2-4) and mutagenesis generation while biasing against the re- subsequent cloning using restriction during PCR by Taq DNA polymerase-me- covery of mutants with multiple muta- sites designed into the primers, re- diated misincorporation. ~s-6) tions. It is, therefore, particularly effec- sults in recovery of a population of The use of separate synthetic oligonu- tive for defining the role of specific bases randomly mutated products. Since cleotides, each with a different defined in DNA sequence elements, by identify- primers with multiple mutations hy- base substitution, is ideal for the isola- ing single base changes that cause a sig- bridize less efficiently to WT tem- tion of a few mutants with specific de- nificant alteration in element function. plate DNA during PCR amplification, sired base changes, such as the creation As with direct cloning of a degenerate the recovery of mutants with multi- of a restriction site or alteration of a spe- oligonucleotide, this method can be eas- ple base changes is greatly reduced. cific amino acid codon. However, it is an ily applied to mutagenesis of a larger el- The efficient generation of random expensive and labor-intensive process ement, for example, a prokaryotic pro- point mutations with this method al- when applied to the saturation mutagen- moter. Prior to saturation mutagenesis, lows the construction of separate esis of even a small, e.g., 10-base, se- the boundaries of such elements are of- mutant populations, each muta- quence. ten defined by deletion analysis, identi- genized over a different portion of Mutagenesis with degenerate oligo- fying the element as a 50- to 150-bp seg- the DNA sequence element. If a phe- nucleotides containing randomly incor- ment, only portions of which may be notypic assay is available, these pop- porated substitutions requires consider- important for function. Important and ulations can be screened directly to ably fewer syntheses. However, cloning unimportant regions can be identified define those regions within the ele- of the mutant oligonucleotides requires quickly by dividing the large element ment that are important for activity. the generation of closely spaced restric- into smaller regions and independently Only those populations containing tion sites within the element or the syn- saturating each small region with ran-

210 PCR Methods and Applications 2:210-217©1993 by Cold Spring Harbor Laboratory ISSN 1054-9803/93 $3.00 Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

I II III IV V VI REGIONS: I il II II 71 II activator footprint GCCGGTTATT TCCTGTCACCATAA~CT GCCACCTGATTTTT~~C CATCAGAGAATT -35 region -10 region +1

~ Degenerate oligonucleotide synthesis •1(15% misincorporation) POOLS OF DEGENERATE OLIGONUCLEOTIDES: Region I primers: EcoR] BamH[ /~.-I I opposing WT primer

/V%l X I /~-; X XXXI e I~1%-; h"" I B /v%;X X I D

Region V primers: BamHI : i x I

= ! .... X X I

| .. | EcoR[ 21 i ^ X~ ,/V% ~, I XX I ,/VN opposing WT primer PCR amplification to produce double- stranded products reduces the frequency of multiple base substitution mutations. MUTANT POPULATIONS: Region I ~', x I /v~j I /vM I ,/V% /vv, x x t /vM x I A/%~ X I JvN

Region V /~/%, I X I /~/%. I /N/% ,I X X I /~/%, 'vX I ~V~ I XI ./vN

Clone and screen mutant populations by phenotypic assays

EXPECTED REGIONAL PHENOTYPES: Proportion of mutant population with indicated phenotype Phenotypically WT Promoter up/down

Region I "unimportant" 97% 3% Region V "important" 45% 55% Further sequencing and - characterization of EXPECTED ANALYSIS: "important" regions Promoter Region V activity G + T ++ A +++ C ++ G ++ A-G FIGURE 1 Strategy for the genetic dissection of a DNA sequence element, the lys promoter of bacteriophage Mu, by mutagenic oligonucleotide- directed PCR.

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dora point mutations. The number and ethanol precipitated, (9) and resuspended Preparation of PCR Templates in 100 ~1 of H20. Degeneracy was intro- degree of phenotypic changes exhibited Template DNA was isolated from Escher- by each regionally distinct population of duced to the positions targeted for mu- ichia coli K-I2 strain MH3016 (F- thyA mutants indicate the importance of each tagenesis by simultaneous delivery of malT::[Mu cts62][P1R P2 R MuR]). ~a°~ nucleotide from two bottles, one with region to activity of the whole element. Briefly, MH3016 was heat induced to the WT nucleotide and a second con- Such a phenotypic screen significantly stimulate Mu replication and thereby in- taining an equimolar mix of all four nu- reduces the work involved in character- crease the copy number of Mu DNA con- cleotides (Applied Biosystems, A/G/C/T izing the mutants because those gener- taining the promoter region. Approxi- cyanoethyl phosphoramidite, 250 mg, ated in unimportant regions need not be mately 10-15 min before lysis, total #400334). The latter bottle was diluted sequenced or characterized further. Se- cellular DNA was isolated by a modified quence analysis of all mutations in the to achieve a total phosphoramidite con- miniprep procedure. ~11'1z) For PCR, 100 important regions, regardless of pheno- centration of 0.025 M, i.e., 6.25 mM of ng of the isolated template was dena- each of the nucleotides, A, G, C, and T. type, will define the relative contribu- tured in a 10-1~1 volume of 0.2 M NaOH Thus, for each targeted nucleotide, syn- tion of individual base pairs to the activ- for 15 rain at room temperature, fol- thesis was accomplished with a mixture ity of the element. lowed by 10-fold dilution into HzO. One of 85% WT and 15% non-WT nude- To be effective for identifying impor- nanogram of denatured template was otides, resulting in a misincorporation tant and unimportant regions, the mu- used immediately in each PCR amplifi- rate of 0.15 per nucleotide. The four oli- tagenesis method must allow the isola- cation reaction. tion of populations that are randomly gomers synthesized were ATGGAATTC- and efficiently mutagenized in the tar- CCGCCGGTTATTI'CCTGTCAC, ATGAA- Amplification Conditions geted region and wild-type in the re- TTCCCgccggttattTCCTGTCACC, CGGG- maining nontargeted bases of the ele- AT C C C CAATTCTCTGATGgcagtctaaaaA- PCR amplifications were carried out in ment. To test the method, we have ATCAG, and ACGGGATCCCCAATTCTC- 100-pA reactions containing Taq DNA applied it separately to two regions TGATGGCAGTCTA, where a lower-case polymerase reaction buffer (10 mM Tris- within a 68-bp promoter. Mutagenesis of letter indicates a position targeted for HC1, pH 8.3, 50 mM KCI, and 0.001% wt/ each region involved two steps: (1) Base mutagenesis. vol gelatin), 1.5-10 mM MgClz maxi- substitution mutations within the targeted region were incorporated during synthesis of a degenerate oligonucleotide to be used as a primer for PCR. (2) PCR amplification with the degenerate primer and an opposing WT primer led to incorporation of the mutant primer and restoration of the remaining WT bases in the 68-bp element. DNA sequence analysis of the mutants demon- strated that this technique is an efficient method for the generation of random AATCGCTAAG ccgagttatg CGAATCGGCC point mutations within the targeted wildtype mutant wildtype DNA segments.

MATERIALS AND METHODS AATCGCTA~ CCGGG_TTATG CGAATCGGCC

AATCGCTAAG CCG~TTATG CGAATCGGCC Oligonucleotide Synthesis AATCGCTAAG CCGAGTTATC CGAATCGGCC Oligomers were synthesized on an Ap- plied Biosystems DNA synthesizer AATCGCTAAG C~TGTG CGAATCGGCC (model 380B) using the phosphite tri- ester method. (z'8) 13-Cyanoethyl phos- AATCGCTAAG CCGAGATATG CGAATCGGCC phoramidites, diluted to 0.1 M in aceto- AATCGCTAAG CCGAGTTCTG CGAATCGGCC nitrile under nitrogen gas to prevent O z inactivation, were 1.0-gram stocks from AATCGCTAAG CCAATTTATG CGAATCGGCC Applied Biosystems (A, #400326, G, #400327, C, #400328, and T, #400329). Pool of oligonucleotides with random base substitutions After ,detachment and removal of pro- at -15% misincorporation tecting groups in 30% ammonium hy- FIGURE 2 Mutagenic oligonucleotide synthesis. Symbols within bottles indicate concentrations droxide at 55°C overnight, the oligomers of specific nucleotide precursors used for synthesis. In the nucleotide sequence lowercase a, g, c, were dried down, resuspended in 200 lxl and t indicate positions targeted for misincorporation; for these positions, nucleotide precursors of Ha0, extracted three times with an from two bottles are added simultaneously. In the resulting products A, G, C, and T indicate the equal volume of HzO-saturated ether, (9) nucleotide present; underlined nucleotides differ from the WT nucleotides.

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mized for specificity and recovery of Sequence Analysis RESULTS amplified product (1.5 mM for Region I, Double-stranded sequencing templates As a test of the method, mutagenesis by and 6 mM for Region V), 200 I~M of each of the mutated promoter clones were degenerate oligonucleotide-primed PCR dNTP, 1 I~M of each of the opposing prepared by a modified Holmes and amplification (Mod-PCR for mutagenic primers, 2.5 units of AmpliTaq poly- Quigley miniprep method involving ly- oligonucleotide-directed PCR) was ap- merase (Perkin-Elmer Cetus), and 1 ng of sis by boiling. ~12'16) The minipreps were plied to two regions within the bacte- denatured template (as described above). alkali denatured in 100 p.1 of 0.2 M NaOH riophage Mu late promoter, Plys. First, After overlaying with 50 ~l of mineral for 30 min at 37°C, neutralized with 40 P1ys was divided into appropriate target oil, the reactions were subjected to 35 i~l of 3 M sodium acetate (pH 4.8), and regions for mutagenesis and screening of cycles of PCR: 1 min at 94°C to denature, ethanol precipitated before being sub- resulting phenotypic changes. Next, a 30 sec at 45°C to anneal, and 2 min at jected to standard US Biochemicals Se- degenerate primer was synthesized for 72°C to extend. The amplification prod- quenase sequencing methods using each target region and used in an inde- ucts were extracted once with CH3CI (24: deoxyadenosine 5'-[~-thio-] triphos- pendent PCR amplification with an op- 1 CH3C1/isoamyl alcohol) to remove the phate, [3SS]- (NEN Research Products) as posing WT primer. Finally, the amplifi- mineral oil, and 5 ~l was subjected to label. Manganese buffer was added to cation products were cloned, assayed for electrophoresis in a 4% 3:1 NuSieve the sequencing reactions according to promoter activity, and sequenced to de- (FMC Bioproducts) minigel to monitor US Biochemicals Sequenase protocols to termine the efficacy of the mutagenesis the amount of product recovered. obtain sequences close to the primer method. (CAGGAATTGGGGATCGG), which was DNA Recovery and Cloning homologous to pLC1 upstream of the Regional Mutagenesis Strategy cloning site. The sequence was visual- The remaining PCR product (-95 ~l af- ized by 6% PAGE in the presence of 8 M Figure 1 illustrates the hypothetical ap- ter minigel analysis) was extracted once urea followed by autoradiography. ~9) plication of regional mutagenesis and with an equal volume of phenol-CH3C1 (saturated with Tris-HCl, pH 8.0) ~9) and ethanol precipitated. The pellet was directly resuspended in 10 i~l of a modified Number ny,une"'"n V Number Region I isolated isolated EcoRIIBamHI restriction buffer (0.15% Triton X-100, 100 mM NaC1, 50 mM Tris- ~:GCCGGTTATT 32 ~:TTTTAGACTGC39 HCI, pH 7.5, 10 mM ~3-mercaptoethanol, and 5 mM MgC12), containing 5 units of C ..... I C 2 A 2 A 3 BamHI (New England BioLabs, Inc.), 2 - T ...... 3 G 1 units of EcoRI (New England BioLabs, - A ...... 2 G 1 - - A ...... 1 - - C ...... 1 Inc.), and 0.1 mg/ml bovine serum albu- - - G ...... 2 - - A 3 min (diluted in H20 from New England - - T ...... 1 A 3 - - A - - - 1 C 1 BioLabs, Inc., 10 mg/ml stock). The di- - - - C ...... 1 G 2 .... A ..... 1 G ...... 2 gestions were carried out for 2 hr at 37°C .... C 1 C 1 or overnight at room temperature. The G .... 3 T 1 G - - - 4 C 1 products were purified in a 4% NuSieve 2 A 1 agarose gel according to FMC Bioprod- C 1 C 1 T - - 2 A 2 ucts' protocols. Ligations were carried G - 1 C 3 out in the melted NuSieve agarose as rec- A- 1 A- - 2 G 3 A - 1 ommended by FMC Bioproducts with C 1 T - 1 pLC1 vector which had been previously A 1 C - 2 digested with EcoRI and BamHI, treated - -A- A ..... 1 C C 1 with calf intestine alkaline phosphatase - A A .... 1 - - C - - A 1 A G - - - 1 C - - - A - 1 (Boehringer Mannheim), and gel puri- - - T - C 1 A .... T 1 fied. Plasmid pLC1 is a lacY derivative of - AG 1 A- G 1 -T- - A- 1 -G T 1 the promoter cloning vector, pRS415, ~1~) C - - C 1 C G 1 generated by deletion of the SnaBI frag- - - TC 1 C- -A 1 C - - - G - 1 A .... C 2 ment in lacY. The ligation mixtures were - -G C 1 A- A 1 transformed into MH8624 (Apro-lac, - - - TA- 2 -A .... C 1 - - - G - G 1 trp~ED24, recA, pLC3) made competent - A .... G .... 1 by CaC12 treatment ~9) and stored frozen. - - T--- GC 1 - - - AC - C 1 Plasmid pLC3 is a monomeric derivative -A C-A- - 1 of pWM13 (14) with the lacIQ cassette -- 87 83 from pMJR1560 ~ls> cloned into the TaqI FIGURE 3 Distribution of base changes recovered in Regions I and V of P~ys. The WT DNA methylase-insensitive HincII site of sequence of the region targeted for mutagenesis is shown at the top. The number of times a pWM13; this plasmid was present to pro- specific sequence was found is indicated on the right. For the mutants, only the mutant nucle- vide an activator necessary for the phe- otides are indicated; A represents a deleted nucleotide; the G in the last Region I double mutant notypic assay of promoter activity. represents an inserted G.

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phenotypic screening to Plys. The 68-bp of all four nucleotides, each at 1/16th the maining WT bases in the 68-bp pro- P~×s promoter would be divided into six concentration of nucleotide in the WT moter were restored by PCR amplifica- 10- to 12-bp regions such that groups of bottle (Fig. 2). Thus, the resulting nucle- tion using a WT primer for the opposing bases expected to be important would be otide mixture in the synthesis chamber end (Fig. 1). After EcoRI and BamHI di- in one region and others predicted to be contained 5% each of the three non-WT gestion, the PCR products were cloned less important would be in a different nucleotides. For a 10-bp mutagenized re- into pLC1, the promoterless lacZ expres- region. Mutant populations for each region, this should result in an average of sion vector, for phenotypic assay and se- gion would be generated by Mod-PCR 1.5 misincorporations (non-WT nucle- quence analysis. and cloned into a promoterless lacZ ex- otides) per oligomer, a level that maxi- pression vector so that an indicator plate mizes the proportion of one- and Randomness and Efficiency of could be used to assay promoter activity. two- base substitutions. This five-bottle Mod-PCR The proportion of clones with WT and synthesis minimizes nonrandom substi- non-WT phenotypes would indicate the tution that can result with the alternate Sequence analysis of clones from the Re- relative contribution of each region to method of preparing separately spiked A, gion I and Region V mutagenized popu- promoter activity. Region V represents a G, C, and T mixes. lations revealed that only 2 of 170 clones region important for activity; many of The degenerate primers were de- contained a mutation outside the tar- the clones in the Region V population signed to contain an overall G + C con- geted regions (a 1-bp deletion and a 1-bp would exhibit an up or down promoter tent of -50%, at least four WT nucle- substitution), confirming the low muta- phenotype. Region I represents a less im- otides on the 3' end to ensure efficient genic activity of PCR itself (-0.08% per portant region in which the majority of priming and, where appropriate, a re- base in our hands for >25,000 bases se- mutants would be phenotypically WT. striction site on the 5' end to allow direct quenced), and demonstrating the fidel- Many clones in the important Region V cloning into the assay vector. The re- ity of restoration of WT sequences population would be sequenced and characterized, regardless of phenotype. For the unimportant Region I, only the TABLE 1 Assessment of Degree of Randomness of Mutagenesis Method clones with altered phenotype would be analyzed further; however, a few pheno- A. Frequency of substitution to non-WT nucleotides typically WT clones, perhaps 10, would be sequenced to demonstrate that phe- Non-WT Number of Mutants Mutants notypically silent mutations were nucleotide substitutions Available expected detected substituted observeda targetb (%)c (%)d present. Thus, the application of Mod- PCR to specific regions of an element A 44 18/63 28.6 35.2 could greatly reduce the number of mu- G 29 16/63 25.4 23.2 tants that need to be sequenced to deter- C 36 17/63 27.0 28.8 mine the bases required for activity. T 16 12/63 19.0 12.8 In this paper, we will present the re- Total 125 sults of sequence analysis of mutants in both Regions I and V (even though Re- B. Frequency of replacement of WT with non-WT nucleotides gion I is "unimportant") because they WT Number of Mutants Mutants represent independent tests of the Mod- nucleotide substitutions Available expected detected PCR method using primers that were replaced observeda targetc (%)e (%)d synthesized with separately prepared nu- A 16 3/21 14.3 12.8 cleotide substrates. The phenotypic G 25 5/21 23.8 20.0 properties of the regionally mutagenized C 21 4/21 19.0 16.8 populations and of specific mutants will T 63 9/21 42.9 50.4 be presented in a subsequent paper. Total 125 Oligonucleotide Synthesis and PCR aThe number of substitutions observed for each class were tallied for substitution to specific non-WT nucleotides (A) and replacement of specific WT nucleotides (B), for all nucleotide An oligomer of -30 nucleotides encom- changes shown in Fig. 2 except deletions and insertions. passing each targeted region was synthe- bThe numerator of the available target for substitution to non-WT nucleotides was determined sized with an automated DNA synthe- by the number of positions targeted for mutagenesis in Regions I and V which were not the sizer. During synthesis of the WT nucleotide indicated. The denominator was defined by the total number of possible substitu- portion of the oligomer, the appropriate tions. In other words, because 21 positions were targeted for mutagenesis in Regions I and V, and at each position 3 non-WT possibilities existed, the total number of possible substitutions was WT nucleotide was added to the synthe- 21 x3 = 63. sis chamber from one bottle. When a nu- CThe expected percentage of each mutant class was calculated from the fraction of available cleotide targeted for mutagenesis was target. reached, equal volumes were added si- dThe detected percentage was the number of substitutions observed in each class divided by the multaneously from two different bottles: total number of substitutions (125). one contained the WT nucleotide; the eThe available target for replacement of WT nucleotide was the number of WT positions of the other contained an equimolar mixture indicated nucleotide among 21 total.

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TABLE 2 Bias Against Multiple Base Substitutions by Mod-PCR Mutagenesis binomial theorem; only 2.4% and 1.2% of the clones in Regions I and V, respec- A. Region I tively, contained more than two substi- Number of changes Expected (%)a Observed (%)b Recovery (%)c tutions (Table 2). In fact, comparison of the observed distribution to that ex- 0 19.7 38.6 100 pected indicates that recovery of the WT 1 34.7 42.2 62.1 class (0 base changes) was significantly 2 27.6 16.9 31.2 favored. The percent recovery by Mod- More 18.0 2.4 6.8 PCR of base substitutions theoretically B. Region V present in the degenerate primer pool was compared to that of the WT class. Number of changes Expected (%)a Observed (%)b Recovery (%)c Relative to WT (100%), one-base substi- 0 16.7 44.8 100 tutions were recovered at 62% and 46%, 1 32.5 40.2 46.1 two-base substitutions at 31% and 18%, 2 28.7 13.8 17.9 and multiple substitutions at 7% and More 22.1 1.2 2.0 2%, for Regions I and V, respectively. These results suggest that primer anneal- aThe percentage of mutants expected in each class (0, 1, 2, or more substitutions) was determined by the binomial theorem with a misincorporation rate of 0.15, and a target size of 10 bp for ing was adversely affected by even one Region I and 11 bp for Region V. mismatch and decreased substantially bThe percentage of mutants observed in each class was determined by sequence analysis of 83 with each additional mismatch present. and 87 mutants in Region I and V, respectively. CThe percent recovery of mutants was calculated relative to 100% recovery of the 0 change (WT) Mutagenesis Strategy for Central class. Region III Mutagenesis of regions at the ends of the within regions not targeted for mutagen- during PCR, the number of mutants re- element, such as I, II, V, and VI of Plys, esis. covered with multiple base substitutions require only one PCR amplification with Figure 3 shows the distribution of (more than two substitutions) was con- the opposing WT primer as diagrammed base changes recovered among 170 siderably less than that predicted by the in Figure 1. Regions in the middle of the clones resulting from Mod-PCR mutagenesis. The isolation of at least one base change at every position targeted II III IV V Vl for mutagenesis was an encouraging sign activator footprint that the mutagenesis was random. In Ta- ble 1 we more systematically investi- I I I I I i gated the degree of randomness by first -35 region -10 region calculating the expected distribution of Region III degenerate oligo specific substitutions based on the target I X ', v Ba,R,U,I size of each base within the mutagenized opposing WT primer sequence. For example, the available tar- (right end primer) get for substitution to A is given by 18 First PCR amplification positions which are not A within the 21 bp targeted for mutagenesis (10 bp of Re- gion I plus 11 bp of Region V). Since the .. I IIIb__~ unuunuu~l~not~nnn~at0n0~un~n~unn~J~a~00~nJtnn~nnun0n~n00n~nnnnnnun~JI l total number of possible non-WT substitutions is 3 non-WT possibilities per position (21x3 = 63), the expected frac- ~ Remove excess primer& target tion of mutations to A should be 18 DNA. Denature strandsand anneal to both end primers. divided by 63, or 28.6% (Table 1A). A similar calculation was done to define iy- letlend primer the expected frequency of mutation IIIIIV from a specific WT nucleotide to any of ~I Ba~l the non-WT nucleotides (Table 1B). Be- right end primer cause the detected distribution of base changes approached that expected for ~ econd PCR amplification. nonbiased misincorporation (Table 1), Only complete promoter is substitution by non-WT nucleotides and exponentially amplified. Region III mutant: replacement of WT nucleotides was es-

sentially the same for A, G, C, or T. •••I••II•••I•I••••••••••••I••••••••I•••••••••••••••••I•III•I••••••••••••••••II••••••••••••I• As expected, due to the requirement for primer hybridization to target DNA FIGURE 4 Recombinant PCR strategy to generate a Region III mutant population.

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element, such as III and IV, are not easily such as temperature, MgC12 concentra- direct cloning of a degenerate oligonu- mutagenized with degenerate oligomers tion, and primer length could be altered cleotide. (2-4) Both methods take advan- that extend to the ends of the element; to fine tune the distribution of single, tage of the ability to target mutations to not only does oligomer cost increase and double, and multiple base substitutions a particular region through the use of yield decrease for longer oligomers, but recovered. In addition, the recovery of synthetic degenerate oligonucleotides, the use of longer primers would be likely WT clones could be reduced by increas- and both can be used to generate a mu- to increase the recovery of the less desir- ing the rate of misincorporation during tant pool containing random, predomi- able multiple mutants. Therefore, mu- degenerate oligonucleotide synthesis, nantly single base mutations. The meth- tagenesis of the central regions would be thereby reducing the proportion of WT ods differ most significantly in the accomplished with a modified recombi- primers. Table 3 illustrates the predicted reduced cost of oligonucleotide synthe- nant PCR method (s~ involving two se- effect of misincorporation rate on the sis for Mod-PCR and the absence of the quential PCR amplifications. For exam- isolation of specific classes of mutants need to introduce closely spaced restric- ple (see Fig. 4), in the first amplification, for the Mod-PCR conditions used here. tion sites into the DNA being muta- the opposing WT primer can be used An increase in the average misincorpo- genized by Mod-PCR. In its application with the degenerate primer to synthesize ration rate from 1.5 to 2.5 misincorpora- to protein structure-function analysis, one end of the element. After gel purifi- tions per primer would halve the num- the direct cloning method has the ad- cation of this short segment to remove ber of WTs recovered in the Mod-PCR vantage that use of a highly degenerate excess primer and target DNA, the re- pool while increasing the number of oligonucleotide can result in the remaining end can be restored by adding double and multiple mutants. Because covery of many different amino acid back WT primers homologous to both the frequency of single mutants would changes at a single position, and the re- ends of the element. The product gener- decrease significantly beyond 2.5 substi- striction sites needed for cloning can be ated from the first amplification would tutions per primer, increased mutagene- introduced without changing the en- contain sequences overlapping with the sis beyond 2.5 would be useful only coded amino acids. In contrast, such a WT primer from the missing end. An- when double and multiple mutants were change in a DNA sequence element may nealing, based on this homology would also desirable. Alternatively, a colony hy- itself alter the activity of the element and prime synthesis of the remaining base bridization assay (17~ with a WT primer as affect the phenotypic properties of the pairs during the second amplification. probe could be used to detect and elim- targeted mutants, thereby resulting in When this two-step recombinant PCR inate WT clones from further analysis; erroneous conclusions. Lastly, the com- was applied to Regions III and IV, it ef- for the rate of misincorporation tested bination of Mod-PCR with recombinant fectively generated the desired mutant (0.15), this would have increased the PCR should allow the application of this populations (data not shown). In cases proportion of single base substitutions method to saturation mutagenesis of in- where the element is considerably to almost 70% of those sequenced. tact wild-type DNA sequence elements longer than 68 bp, multiple independent Mod-PCR should provide an effective too long to be mutagenized by the direct PCR products that overlap at their ends complement to the existing method of cloning method. could be synthesized and combined with outside primers to generate the intact element. TABLE 3 Expected Effect of Misincorporation Rate During Mutagenic Oligonucleotide Synthesis on Mutant Recovery Following Mod-PCR

DISCUSSION Distribution in oligo pool b Distribution in Mod-PCR pool c Rate a 0 1 2 more 0 1 2 more The results presented here indicate that Mod-PCR is a convenient method for sat- 0.100 0.31 0.38 0.21 0.10 0.54 0.36 0.09 0.01 uration mutagenesis of a DNA sequence 0.125 0.23 0.36 0.26 0.15 0.46 0.40 0.13 0.02 element. Primer synthesis with the WT 0.129 0.22 0.36 0.26 0.16 0.45 0.40 0.14 0.02 and appropriately diluted equimolar 0.150 0.17 0.32 0.29 0.22 0.39 0.42 0.17 0.03 mixture of nucleotide substrates resulted 0.175 0.12 0.28 0.30 0.30 0.33 0.42 0.20 0.04 in the generation of random base substi- 0.200 0.09 0.24 0.30 0.38 0.28 0.42 0.24 0.06 0.225 0.06 0.20 0.28 0.46 0.23 0.41 0.27 0.09 tution mutations within the targeted re- 0.250 0.04 0.16 0.26 0.54 0.19 0.39 0.29 0.12 gion. Annealing conditions during Mod- 0.275 0.03 0.12 0.23 0.62 0.16 0.36 0.31 0.17 PCR effectively reduced the recovery of 0.300 0.02 0.09 0.20 0.69 0.13 0.33 0.32 0.22 mutants containing more than two base 0.325 0.01 0.07 0.17 0.75 0.10 0.29 0.32 0.28 changes. Thus, Mod-PCR can be effi- 0.350 0.01 0.05 0.14 0.80 0.08 0.26 0.31 0.35 ciently applied to independent satura- 0.375 0.01 0.04 0.11 0.84 0.06 0.22 0.29 0.43 tion mutagenesis of distinct regions aMisincorporation rate during mutagenic oligonucleotide synthesis, adjusted by altering the within a DNA sequence element, first de- concentration of nucleotide precursors in the equimolar mixture bottle. fining the regions, and then the bases, bDetermined by the binomial theorem for the indicated misincorporation rate and an ll-nucle- important to element function. otide region targeted for mutagenesis. Although not tested in this paper, we CBased on Mod-PCR conditions that result in 100% recovery of the WT class, 55% recovery of the would presume that parameters affect- single mutant class, 25% for double mutants, and 5% for multiple mutants (average recovery ing annealing conditions during PCR, rates observed for Regions I and V rounded to the nearest 5%).

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ACKNOWLEDGMENTS 1992. DNA sequence characterization of the G gene region of bacteriophage Mu. This work was supported by the College DNA Sequence 2: 329-334. of Medicine, The University of Tennes- 13. Simons, R.W., F. Houman, and N. Kleck- see -- Memphis, by National Science ner. 1987. Improved single and multicopy Foundation grant DMB 9006364 to lacobased cloning vectors for protein and M.M.H., and by a University of Tennes- operon fusions. Gene 53: 85-96. see Van Vleet Professorship. Oligonucle- 14. Margolin, W., G. Rao, and M.M. Howe. otides were provided by the Molecular 1989. Bacteriophage Mu late promoters: Resource Center, The University of Ten- Four late transcripts initiate near a con- served sequence. J. Bacteriol. 171: 2003- nessee -- Memphis. The authors thank J. 2018. Swindle for advice on PCR and com- 15. Stark, M.J.R. 1987. Multicopy expression ments on the manuscript, and C.A. Baxa vectors carrying the Lac repressor gene for for providing template DNA and modi- regulated high-level expression of genes fied protocols for sequencing and chro- in Escherichia coli. Gene 51: 255-267. mosomal minipreps. 16. Holmes, D.S. and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193-197. REFERENCES 17. Ausubel, F.M., R. Brent, R.E. Kingston, 1. Smith, M. 1985. In vitro mutagenesis. D.D. Moore, J.G. Seidman, J.A. Smith, and Annu. Rev. Genet. 19: 423-462. K. Struhl. 1991. Current protocols in molec- 2. Hill, D.E., A.R. Oliphant, and K. Struhl. ular biology. John Wiley & Sons, New 1986. Mutagenesis with degenerate oligo- York. nucleotides: An efficient method for saturating a defined DNA region with base pair substitutions. Methods Enzymol. 155: 558-568. 3. Bowie, J.U. and R.T. Sauer. 1989. Identify- ing determinants of folding and activity for a protein of unknown structure. Proc. Natl. Acad. Sci. USA. 86: 2152-2156. 4. Dube, D.K. and L.A. Loeb. 1989. Mutants generated by the insertion of random oligonucleotides into the active site of the 13-1actamase gene. Biochemistry 28: 5703- 5707. 5. Higuchi, R. 1989. Using PCR to engineer DNA. In PCR Technology (ed. H.A. Erlich), pp. 61-70. Stockton Press, New York. 6. Leung, E.W., E. Chen, and D.V. Goeddel. 1989. A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1: 11-15. 7. Matteucci, M.D. and M.H. Caruthers. 1981. Synthesis of deoxynucleotides on a polymer support. J. Am. Chem. Soc. 103: 3185-3191. 8. Beaucage, S.L. and M.H. Caruthers. 1981. Deoxynucleoside phosphoramidites -- a new class of intermediates for deoxypoly° nucleotide synthesis. Tetrahedron Lett. 22: 1859-1862. 9. Maniatis, T., E.F. Fritsch, andJ. Sambrook. 1982. Molecular cloning: A laboratory man- ual. Cold Spring Harbor Press, Cold Spring Harbor, New York. 10. Grundy, F.J. and M.M. Howe. 1984. In- volvement of the invertible G segment in bacteriophage Mu tail fiber biosynthesis. Virology 134:296-317. 11. Schleif, R.F. and P.C. Wensink. 1981. Prac- tical methods in molecular biology. Springer-Verlag, New York. 12. Baxa, C.A., L. Chiang, and M.M. Howe.

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Mutagenic oligonucleotide-directed PCR amplification (Mod-PCR): an efficient method for generating random base substitution mutations in a DNA sequence element.

L W Chiang, I Kovari and M M Howe

Genome Res. 1993 2: 210-217 Access the most recent version at doi:10.1101/gr.2.3.210

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