Quick viewing(Text Mode)

DNA Polymerase Fidelity and the Polymerase Chain Reaction Kristin A

DNA Polymerase Fidelity and the Polymerase Chain Reaction Kristin A

Downloaded from .cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

DNA Fidelity and the Polymerase Kristin A. Eckert and Thomas A. Kunkel

Laboratory of Molecular , National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709

The distinctive ability of the because errors generated during can be controlled experimentally. polymerase chain reaction (PCR) to amplification may interfere with the produce a substantial quantity of DNA interpretation of data. ERROR ACCUMULATION DURING from an initially small amount of ge- During enzymatic DNA amplifica- PCR netic material is revolutionizing tion, the majority of changes in nucle- Error rates in PCR vary according to . The variety of high- otide sequence can be attributed to er- the precise DNA sequence and the in ly successful applications of PCR tech- rors made by the DNA polymerase. vitro conditions of DNA synthesis. nology and the ease with which PCR While PCR is a recent development in Several laboratories have estimated the can be performed should not be the field of molecular biology, the fi- error frequency (mutations per misinterpreted as evidence that the delity of DNA has been per cycle) during PCR PCR is a simple process. In fact, quite studied biochemically for almost 30 catalyzed by the thermostable Thermus the opposite is true. A single cycle of years. In this review, we will attempt to aquaticus (Taq) DNA polymerase by DNA polymerization is a complex pro- relate our current understanding of the cloning individual DNA cess requiring the precise interaction of mechanisms of polymerase fidelity in from the amplified population and several components. The repetitive vitro to the accuracy of enzymatic determining the number of DNA se- cycles characteristic of PCR provide an DNA amplification. We begin by de- quence changes. (6-12) As seen from the additional layer of complexity to the scribing steps in polymerase error dis- data in Table 1, observed error frequen- technique. The final PCR product can- crimination and variables important cies during PCR vary more than 10- not be considered a unique entity, as for determining fidelity. The error rates fold, from -2 x 10 -4 to <1 x 10 -5 . For even a discrete DNA band on an of several DNA polymerases that have applications of PCR products that util- agarose gel may contain a variety of been used for PCR will be described, ize the population of amplified DNA DNA molecules differing from the first as determined in model "one- molecules, random errors at these fre- original genetic information by one or cycle" fidelity assays and then during quencies will not interfere with the more . actual PCR. Our intent is to focus on biochemical analyses. However, poly- How serious a concern is the fidel- the variables known to influence the merase-mediated errors at a frequency ity of PCR? The answer depends on the fidelity of DNA synthesis in vitro that of 1 mutation per 10,000 nucleotides precise nature of the application. In many instances, for example, direct characterization of the amplified popu- lation by DNA sequence analysis or TABLE 1 Estimates of Fidelity during PCR Catalyzed by hybridization, random er- rors in nucleotide sequence that may Error Error rate Target Number frequency per nucleotide be produced during PCR are of little sequence of cycles per nucleotide per cycle a Reference concern. However, some PCR applica- tions involve the characterization of Apolipoprotein B 30 22/8,000 1/5,600 6 individual DNA molecules or rare HLA-DPf~ 30 17/6,692 1/5,900 7 molecules present in a heterogeneous R-Antit rypsin 33 7/4,700 1/ 11,000 8 population. Examples include the HPRT 32 16/15,000 1/15,O00 9 study of allelic polymorphism in indi- HLA-A,B 30 20/21,870 1/16,000 10 vidual mRNA transcripts O,2) and the 20 17/30,710 1/18,000 10 characterization of the allelic states of TCR V8 25 1/1,500 1/19,000 11 single sperm cells (3) or single DNA HIV gag/env 30 < 1/5,411 < 1/83,0OO 12 molecules. (4,s) In these circumstances, the fidelity (error rate per nucleotide) aError rate = I/[(observed error frequency/number of cycles) x 2], assuming a constant ef- of PCR is an important consideration, ficiency per cycle. U)

1:1 7-249 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/91 $3.00 PCR Methods and Applications 1 7 Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

per cycle are an important considera- larger than average number of purified polymerases in vitro increase tion for any PCR application that be- . ~ls) The remainder of this as the length of the repeated sequence gins with a small amount of starting review explores the ways in which the increases. (16) Alternative models have material (i.e., less than a total of polymerase error rate, p, can be also been proposed, including dele- 10,000 nucleotides of target DNA) or manipulated experimentally as a tions initiated by base misinsertion. (19) that focuses on individual DNA means of controlling the PCR error fre- Substantial evidence from model as- molecules present in the final PCR quency. says demonstrates that polymerases population. also can generate deletions of a few to The proportion of DNA molecules MOLECULAR NATURE OF hundreds of nucleotides during that contain sequence changes is a MUTATIONS polymerization in vitro. These dele- function of the error rate per nucleo- A variety of changes in DNA sequence tions often involve directly repeated tide per cycle, the number of amplifi- can occur during enzymatic DNA sequences and palindromic or quasi- cation cycles, and the starting popula- amplification. These mutations vary palindromic sequences that have the tion size.t13,14) The population of from single base-substitution muta- potential to form stern-and-loop DNA altered DNA molecules arises during tions to deletions and insertions. 16-12) structures.~177 PCR from two sources: (1) new errors The most familiar type of mutation is In addition to intramolecular errors at each cycle and (2) the amplification the base substitution that results from during DNA synthesis, deletions and of DNA molecules containing errors the misincorporation of an incorrect other types of rearrangements can oc- from previous cycles. On average, the dNTP during DNA synthesis. Twelve cur between DNA molecules. Such in- same number of DNA sequence distinct base mispairs are possible, termolecular events are thought to changes in the final population results varying in composition and in the arise when the polymerase terminates from polymerase errors during the last template base versus the incoming synthesis on one DNA strand and con- amplification cycle as result from the dNTP (e.g., T'dGTP versus G'dTFP). tinues synthesis after priming occurs amplification of errors that occurred Together with the effects of neighbor- on a complementary DNA strand during previous cycles. The formula f = ing template sequence, this creates a (strand switching or jumping PCR). (2~ np/2 describes the average mutation variety of possible molecular structures Both the presence of a high degree of frequency (f) for PCR amplification as a among which any polymerase must sequence homology between regions function of the polymerase error rate discriminate. All DNA polymerases ex- of DNA molecules and a high DNA per nucleotide per cycle (p) and the amined to date also generate muta- concentration increase the incidence number of cycles (n), assuming that p tions during DNA polymerization in of this type of error in PCR. (1~176 is constant at each cycle (for more vitro wherein one or more nucleotides detailed mathematical analyses, see is lost or added. One factor that will in- ERROR DISCRIMINATION BY DNA refs. 13 and 14). Theoretically, the fre- fluence the types of DNA sequence POLYMERASFS AND FIDELITY IN quency of DNA sequence changes can changes (base substitution versus dele- MODEL SYSTEMS be controlled by altering the number tion mutation) likely to occur during Since the majority of DNA sequence of cycles (n) and/or the polymerase er- enzymatic amplification is the se- changes introduced during PCR may ror rate per nucleotide (p) (see Table 6 quence of the target DNA. be polymerase-mediated, we will in ref. 13). For example, as the number The frequency of deletion muta- review what is known about DNA of cycles increases, the error frequency tions is sequence dependent and is in- polymerase error rates from model in is expected to increase. Keeping p con- creased in repetitive DNA sequences vitro systems. Much of our under- stant at one error per 10,000 (for reviews, see refs. 16 and 17) Many standing of fidelity is derived from as- nucleotides per cycle, the error fre- examples exist for the repetitive nature says that quantitate errors made by quency when n = 20 cycles is one of DNA, from simple reiteration of a DNA polymerases during synthesis change per 1000 nucleotides and in- single base, to alternating dinucleotide equivalent to a single PCR cycle. (21-24) creases to one change per 400 tracts in potential Z-DNA structure, to Among these, the broadest description nucleotides after 50 amplification short direct repeats. The generation of of fidelity comes from the M13mp2 cycles. Alternatively, the final error fre- deletion mutations is likely to be forward mutation , (24) which quency can be varied by changing p, determined by the stability of the detects all possible base substitution er- the polymerase error rate. Keeping a mutational intermediates.(16,17) The rors along with a variety of base dele- constant cycle number of n = 50, the most common polymerase-mediated tion and addition errors at a large final mutation frequency is only deletion error is the loss of one number of sites. In this assay, single- 1/2000 nucleotides if p = 1/50,000, but nucleotide. As first proposed by stranded M13mp2 DNA containing the increases to 1/400 nucleotides if p = Streisinger, (18) one mechanism for the c~-complementation region of the Es- 1/10,000. Due to the exponential na- gain or loss of a single nucleotide is the cherichia coli lacZ is used as the ture of PCR, the occurrence of an early misalignment within a repetitive template for a single cycle of DNA error can increase the final error fre- homopolymeric sequence of the DNA synthesis. Upon transfection of an ap- quency above the average described by template-primer during synthesis. propriate E. coli strain with the pro- f = np/2, because the DNA Consistent with this mechanism, the ducts of the reaction, accurate DNA molecules will be amplified with each error rates for some minus-one-base synthesis can be detected as dark blue cycle, resulting in populations with a deletion mutations produced by M13mp2 plaques. However, -

18 PCR Methods and Applications Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

ase errors during synthesis of the lacZ error discrimination is the more rapid ate (step D). The probability of preserv- gene result in lighter blue or colorless rate of formation ing any mispaired intermediate as a plaques, due to decreased cz-comple- for correct rather than for incorrect mutation is increased by reaction con- mentation of 15-galactosidase activity in E'DNA~ complexes (step B). Ob- ditions that increase the rate of exten- the infected host cell. The error rates servations from several laboratories sion synthesis, such as high dNTP con- for specific classes of polymerase errors suggest that DNA polymerases use se- centrations, high concentra- during a single synthesis cycle can be lectivity at both steps A and B to im- tions, or long incubation times. The calculated from the frequency of light prove base substitution fidelity, but rate of mispair extension varies for blue and colorless (mutant) plaques that the relative contribution of the both the type of mispair and the among the total plaques scored and two steps varies from one polymerase polymerase. (32,33) In general, purine- DNA sequence analysis of a collection to the next. (26-28) purine mispairs are the most difficult of the mutants. A more sensitive assay Once the phosphodiester bond is to extend (10 -s to 10-6), while for specific types of mutations can be formed between the incoming dNTP purine-pyrimidine and pyrimidine- performed by using an M13mp2 and the DNA primer, the rate of pyrimidine mispairs are extended more template DNA which contains a base release (step C) may oc- efficiently (10 -2 to 10 -4 ) as compared substitution or deletion mutation, cur more slowly in the case of incorrect to the corresponding correct base pairs resulting in a colorless phenotype. In incorporations. (21) This third level of (M. Goodman, pers. comm.). For the these assays, polymerase errors are discrimination, together with the ob- Taq polymerase, the failure to extend scored as DNA sequence changes that servation that some DNA polymerases mispairs is not due to differential bind- revert the mutant back to a wild-type catalyze pyrophosphorolysis and ing of the polymerase tO correct versus or pseudo-wild-type phenotype. This pyrophosphate exchange reactions, (29) incorrect terminal base pairs, but rath- approach is especially useful for highly provides the possibility that the er is due to a kinetic block to the addi- accurate polymerases. Unlike the for- presence of pyrophosphate in a tion of the next nucleotide onto the ward mutation assay, however, polymerization reaction may influence mispair (M. Goodman, pets. comm.). M13mp2 reversion assays are focused fidelity. Both mutagenic (3~ and anti- As for other DNA polymerases, the in- on a limited subset of errors occurring mutagenic(3]) pyrophosphate effects trinsic extension efficiency for Taq at only a few sites. on the fidelity of E. coli DNA polymer- polymerase measured under steady- Polymerase-mediated discrimina- ase I have been observed. state conditions differs for each type of tion against errors during synthesis oc- A fourth level of error discrimina- mispair (M. Goodman, pers. comm.). curs at several steps in the biochemical tion is the selective ability of a Finally, one of the most important reaction (Fig. 1) (for review, see ref. polymerase to continue DNA synthesis control points for the production of 25). The first control point is the bind- on a correct primer-template structure mutations is the 3' ~5' exonucleo- ing of the dNTP substrate to the poly- rather than on a mutational intermedi- lytic removal () of 3' ter- merase (step A). The rate of binding of both the correct and incorrect incom- ing deoxynucleoside triphosphate is as- A sumed to be the same. However, the E+DNA ~ E~ n -~---~--~ E.DNAndNTP correct dNTP substrate may remain n preferentially bound to the enzyme- dNTP DNA complex due to - ing and base-stacking interactions. B Alternatively, an incorrect dNTP may dissociate from the E~ com- PP plex more readily than a correct i dNTP. (21,22) At this stage, polymerase error rates can be varied experimental- E'DNA,I ~ E.DNA" PP ly by adjusting the ratio of correct and E~ "~ C n+l i incorrect dNTP substrates, reflecting the relative probability that a polymerase will bind an incorrect dNMP versus a correct dNTP. An example of E.DNA EoDNA such a reaction catalyzed by Taq n n polymerase is shown in Table 2 (part FIGURE 1 Steps in polymerase error discrimination. This reaction scheme for error dis- I), wherein base substitution fidelity is crimination by DNA polymerases in vitro has been generalized from data for a varietv of en- decreased eightfold by decreasing the zymes, (2s) but relies heavily on data generated using the of E. coli DNA concentration of a single correct polymerase I. (21) (E) Polymerase; (DNAn) starting DNA primer-template of length 17; nucleotide. Imbalances in the dNTP (DNAn+I) DNAn primer-template after incorporation of one nucleotide; (DNAn+2) DNAn§ 1 pools can be either mutagenic or primer-template after extension synthesis; (dNTP) deoxynucleoside triphosphate; (PPi) antimutagenic, depending on the error pyrophosphate; (dNMP) deoxynucleoside monophosphate. Letters A-E denote control points being considered. A second point for for error discrimination (see text for details).

PCR Methods and Applications 19 Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

TABLE 2 Comparative DNA Polymerase Fidelity in the M13mp2 Mutation Assays tive T7 polymerases can have an ex- quisitely high fidelity for particular Base substitution base-substitution errors, the fidelities 3 ' --,5 ' error rate per are significantly lower when a larger Polymerase nucleotide a Reference number of sites and classes of errors are I. Taq - 1/2,400 b 13 monitored in a forward mutation as- Taq (100 pM dNTP) - 1/290 b 13 say. Given that thermostable DNA II. Klenow (1 pM dATP) + 1/110,000 35 polymerases are preferred for PCR, it is Klenow (1 uM dNTP) - 1/15,000 35 noteworthy that preparations of the III. T4 + 1/300,000 T.A.K. (unpubl.1 thermostable DNA polymerase from 1,80,000 c 13 (Vent polymer- Native T7 + 1/290,000 T.A.K. (unpubl. } ase) contain a 3'~5' exonuclease ac- 1/53,000 c 13 tivity (P. Mattila and J. Ronka, pers. comm.). These investigators have Klenow + 1/45,000 50 demonstrated that this enzyme has a 1/27,000 c 13 base-substitution error rate of 1/31,000 T. litoralis + 1/31,000 P. Mattila Ipers. comm.) in reactions containing 1 mM dNTPs, IV. AMV (500 ~M dNTPs) - 1/24,000 51 similar to that observed for the Klenow Taq - 1/5,600 13 polymerase (Table 2, part liD. Con- Sequenase 2.0 - 1/5,000 c T.A.K. (unpubl.) sistent with the possibility that ex- onucleolytic removal of base- V. Taq (4 mM Mg 2+) - 1/300,000 37 substitution errors is occurring during Taq (pH 5.7) - 1/180,000 37 polymerization, the fidelity of this All fidelity measurements were done using 1 mM each of four dNTPs and 10 m.M .MgC12 un- polymerase is fivefold higher than the less otherwise indicated by parenthetical notations. The T4, native T7, Sequenase 2.0. AMV. fidelity of the exonuclease-deficient and Klenow polymerase reactions were incubated at 37~ the Taq polymerase reactions at Taq polymerase under identical reac- 70~ and the T. litoralis polymerase reactions at 72~ tion conditions (Table 2, parts III and aExcept where noted, error rates per detectable nucleotide were determined from the mutant IV), and the 3' ~5' exonuclease ac- frequency observed in an opal codon reversion assay as previously described. ~~2~ tivity has a slight preference for exci- sion of mismatched rather than bThe base substitution error rate was calculated for changes at the first position tl'G~ of the matched base pairs (P. Mattila and J. TGA codon only. As determined by DNA sequence analysis, first position changes account Ronka, pers. comm.). for 85% of the total errors produced by Taq polymerase at equal dNTP concentrations. The majority of commercially avail- CThe base substitution error rate was calculated as described (50) from DNA sequence analysis able thermostable DNA polymerases do of mutants produced in the M13mp2 forward mutation assay. not contain 3'~5' exonuclease ac- tivities. When the fidelities of various polymerases are compared using minal mispairs to regenerate the start- excision (step E). This balance is not similar reaction conditions, the Taq, ing DNA configuration (step E) (for the same for all polymerases, as (Tth), and review, see ref. 34). Slow pyrophos- demonstrated by the observation that Thermus flavis (Replinase) DNA poly- phate release after an incorrect in- the T4 and native T7 DNA polymerases merases are approximately lO-fold less corporation (step C) and slow exten- are more accurate than is the Klenow accurate than are the T4 or native T7 sion from a mispaired terminus (step polymerase under identical reaction polymerases, consistent with these en- D) increase the opportunity for selec- conditions (Table 2, part Ill). Further- zymes being proofreading-deficient tive removal of misincorporated nucle- more, this balance can be modulated (Table 2) (13) (P. Mattila and J. Ronka, otides by the exonuclease activity. A by changing the conditions of in vitro pers. comm.). Even among exonucle- direct comparison of the 3'---,5' polymerization. For example, proof- ase-deficient DNA polymerases, error exonuclease-proficient versus exo-nu- reading by the Klenow polymerase is rates are not constant. The fidelity of clease-deficient form of Klenow poly- less effective at 1 mM dNTPs than at 1 the AMV is signifi- merase (Table 2, part II) shows a seven- pM dNTPs (Table 2, parts II and Ill); the cantly higher than the fidelities of the fold difference in the base substitution high substrate concentration increases Taq polymerase or the exonuclease- error rates of these , reflecting the rate of extension from mispaired deficient form of T7 polymerase (Se- the contribution of proofreading to the termini (step D), lessening the time quenase 2.0) (Table 2, part IV). Fluctua- fidelity of this polymerase. (3s) available for excision of the error (step tions in base-substitution error rates of The efficiency of proofreading as an E). Table 2 (part III) also illustrates that more than 10-fold have been seen with error discrimination mechanism re- the overall fidelity of a polymerase several DNA polymerases, reflecting flects a balance between the compet- reaction is controlled by the least ac- differences in polymerase error dis- ing processes of polymerization of the curate reaction that occurs. Although crimination at steps A-D (Fig. 1), the next correct nucleotide (step D) and the proofreading-proficient T4 and na- molecular nature of the error, and the

20 PCR Methods and Applications Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

surrounding DNA sequence (for a more of to produce concentrations decrease polymerase fi- extensive discussion, see ref. 36). uracil. (13,38) Since uracil has the same delity by decreasing the amount of er- Not unexpectedly, the fidelity of a coding potential as , faithful ror discrimination at the extension polymerization reaction in vitro replication of a template uracil by a step, while low dNTP concentrations depends upon the synthesis conditions DNA polymerase will result in a increase fidelity by reducing the rate of chosen. The influence of variations in C'G~T~ transition mutation. In PCR, mispair extension. Such error dis- reaction conditions on the fidelity of the greatest risk of cytosine deamina- crimination at the extension step can Taq polymerase has revealed that even tion is during the denaturation step. be utilized for primer-specific PCR this exonuclease-deficient DNA poly- The rate constant for deamination of amplification.(41,42) Thus, template- merase can be highly accurate. Sig- cytosine in single-stranded DNA at primers can be differentially extended nificant improvements in fidelity 80~ is -1 x 10 -8 events/sec, increasing during PCR, depending upon the pre- resulted from decreasing the MgC12 to -2 x 10 .7 events/sec at 95~ A sec- cise nature of the 3' terminal concentration or the pH of the reac- ond common type of DNA damage is mispair. (41,43) While primers creating a tion. (37) Under these conditions, the spontaneous base release resulting T'G (primer-template), G'T, C'A, or error rate per nucleotide polymerized from hydrolysis of the N-glycosylic A'C mismatch provide efficient ampli- at 70~ can be as low as 10 -s for base- bond. (39) The rate of this hydrolytic fication, (41) the presence of an A'A substitution errors, similar to the ob- reaction is significantly increased at mismatch reduces synthesis approxi- served error frequencies of proof- high temperatures and low pH. Depur- mately 20-fold, and the presence of reading-proficient enzymes (Table 2, ination of native DNA at pH 7.4 and A'G, G'A, or C'C mismatches reduces part V). In these studies, fidelity was 70~ occurs at a rate of -4 x 10 -9 events overall PCR product yield about 100- highest at a MgCI 2 concentration that per sec. The release of pyrimidines, fold relative to amplification from cor- was equimolar to the total concentra- while approximately 20 times slower rectly base-paired primers. (43) In addi- tion of dNTP substrates present in the than purines, increases significantly as tion, when the dNTP concentration is reaction, and fidelity decreased as the the temperature is increased to 95~ lowered from 800 i.tM to 6 BM, only T~ concentration of free divalent cation The rate of spontaneous purine loss in mismatches or perfect base pairs are increased. (37) Low rates for both base- native DNA is markedly increased at extended.(43) substitution and minus-one base dele- low pH. Following spontaneous base Recently, Keohavong and Thilly tion errors are also obtained when Taq hydrolysis, the resulting apurinic/ have described a method to quantitate polymerase reactions were carried out apyrimidinic (AP) sites in the DNA can errors produced during PCR using using Tris, 4-morpholineethane-sulfon- inhibit synthesis by DNA polymer- denaturing gradient gel electrophoresis ic acid (MES), and 1,4-piperazinebis ases. (39) In some cases, DNA polymer- (DGGE). (44) In this technique, a 224-bp (ethane-sulfonic acid) (PIPES) buffers at ases are capable of replicating past AP DNA fragment containing the HPRT pH 5-6 (70~ The base-substitution sites. As AP sites are noncoding lesions, exon 3 sequence is amplified by PCR. error rate increased 60-fold as the pH such bypass replication is error-prone After purification of the desired DNA was raised approximately three and results in base-substitution muta- band by polyacrylamide gel electro- units) 37) The fidelity of the Taq tions, most frequently transversions. phoresis, the DNA strands are dena- polymerase is moderately temperature The presence of DNA damage also tured and allowed to reanneal in solu- dependent. (13) Two- to threefold in- increases the occurrence of jumping tion, generating heteroduplex mole- creases in both base-substitution and PCR. The amount of intermolecular cules between DNA strands containing minus-one base deletion error rates DNA rearrangements relative to intra- errors (mutant) and wild-type DNA were observed as the reaction tempera- molecular amplification during PCR strands. A linear gradient of denatur- ture was increased from 23-30~ to was found to be greater when the start- ant in a polyacrylamide gel is then 70-80~ Finally, the overall error rate ing DNA contained double-stranded used to separate the mutant/mutant or of Taq polymerase was observed to in- breaks, AP sites, or light mutant/wild-type heteroduplexes from crease as the dNTP concentration was photoproducts. (2~ Strand-switching the wild-type homoduplex molecules. increased from 1 gM tO 1 mM. (13,37) becomes an important consideration The proportion of mutant DNA mole- This suggests that the fidelity of PCR to fidelity when ancient DNA or DNA cules in the population is estimated amplification can be improved using samples from forensic analyses are from either the heteroduplex frac- low dNTP concentrations, since un- used as the original DNA for amplifica- tion (44'46) or from the wild-type homo- extended mispairs will be lost during tion, because these samples often con- duplex fraction.(4s) Finally, the poly- PCR because they do not yield full- tain various forms of DNA damage. (4~ merase error frequency per length DNA products for further per cycle is calculated from the mutant amplification. ERROR DISCRIMINATION DURING fraction, the number of duplications, PCR and the DNA target size. If desired, in- DNA DAMAGE IN PCR Generally, the rate of polymerase ex- dividual heteroduplex bands can be ex- Incubation of DNA at high tempera- tension from mispaired or misaligned cised to determine the exact nature of tures during PCR produces DNA primer-termini (Fig. 1, step D) is de- the mutation. Unlike the M13mp2 fi- damage, thus increasing the potential pendent upon the concentration of the delity assay, which measures errors for mutations. One of the most fre- next correct dNTP substrate. (32) In the produced during a single round of quent forms of DNA damage is absence of proofreading, high dNTP DNA synthesisJ 24) the DGGE/PCR

PCR Methods and Applications 21 Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

technique detects the polymerase er- in the M13mp2 system. Unexpectedly, repetitive in nature may pose special rors that accumulate during repeated the high fidelity observed during PCR problems for PCR amplification. cycles of amplification. In this system, by Sequenase requires high concentra- Strand-switching events between ho- only those errors that allow full-length tions of deoxynucleoside triphos- mologous DNA segments have been synthesis are recovered in the final phates, and the error rate increases up observed during PCR amplification of population, whereas errors that inhibit to 1/5000 when <0.5 mM dNTPs are HLA genes, and become more preva- extension synthesis will be lost. DGGE used. (46) The reason for this depen- lent during the final cycles of amplifi- has been used successfully to dence is as yet unknown. cation when the concentration of DNA quantitate the error frequency of PCR The influence of PCR conditions on becomes high. (1~ An increased fre- catalyzed by various polymerases un- the fidelity of Taq polymerase was also quency of spurious DNA products with der differing conditions at the HPRT examined using the DGGE/PCR tech- cycle number was also observed during exon 3 target locus (Table 3). (44-46) nique. (46) In these studies, less extreme amplification of tandemly repeated The fidelity of PCR catalyzed by the synthesis conditions could be used as minisatellite DNA. (48) Attempts to Taq polymerase as measured by DGGE compared to the M13mp2 fidelity as- amplify a segment of the human 18S is -1-2 • 10 -4 (Table 3), in close agree- says, (37) since suboptimal reaction con- rRNA gene using the Taq polymerase ment with the estimates from cloning ditions resulted in a decreased ef- generated a 54-bp deletion artifact con- PCR products (Table 1) and with the fi- ficiency or absence of amplification. sistent with the formation of a stable delity of Taq polymerase in M13mp2 Nevertheless, Taq polymerase fidelity intrastrand hairpin loop. (49) Inter- in vitro assays (Table 2). Higher-fidelity was observed to decrease as the reac- estingly, this deletion was not ob- PCR was obtained using the proof- tion pH was increased from pH 8 served when the PCR amplifications reading-proficient T4 DNA polymerase (25~ to pH 9 (25~ and to decrease were performed using the modified T7 (-1/100,000) or the thermostable as the dNTP concentration was in- polymerase, illustrating again that Thermococcus litoralis DNA polymerase creased from 50 [xM tO 200 [xM.(46) Sur- DNA polymerases can differ signifi- (Table 3). PCR catalyzed by the chemi- prisingly, the highest fidelity was ob- cantly in their biochemical properties. cally modified form of T7 DNA poly- served at still higher dNTP concentra- merase (Sequenase) yielded an error tions. At 500 BM dNTPs and 5 mM SUMMARY frequency of approximately one error Mg 2+, the observed error rate was High-fidelity DNA synthesis conditions per 25,000 nucleotides per cycle (Table 1/14,000; however, amplification un- are those that exploit the inherent 3). These observations do not conflict der these conditions was inefficient ability of polymerases to discriminate with low-fidelity DNA synthesis ob- and generated several products other against errors. This review has de- tained with Sequenase 2.0 in the than the desired 224-bp HPRT frag- scribed several experimental ap- M13mp2 assay (Table 2). The differ- ment. (46) The fidelity of the Thermo- proaches for controlling the fidelity of ence presumably reflects the fact that coccus litoralis polymerase is constant enzymatic DNA amplification. One of the chemically modified T7 polymer- over a broad range of dNTP concentra- the most important parameters to con- ase retains a low but detectable 3' --,5 ' tions (10 BM to 200 ~M).(4s) However, sider is the choice of which polymerase exonuclease activity, while Sequenase when 1 mM dNTPs were used for to use in PCR. As demonstrated by the 2.0, a 28-amino-acid deletion mutant, synthesis, the error rate of T. litoralis data in Tables 2 and 3, high-fidelity is completely devoid of exonuclease ac- increased to 1/10,000, (46) consistent DNA amplification will be best tivity. (47) The disparity may also reflect with possible inhibition of proofread- achieved by using a polymerase with the selective loss during PCR amplifica- ing activity at high dNTP concentra- an active 3 ' ----5 ' proofreading ex- tion and/or DGGE analysis of some tion.(34) onuclease activity (Fig. 1E). For those types of mutations that can be scored DNA target sequences that are enzymes that are proofreading- deficient, the in vitro reaction condi- tions can significantly influence the TABLE 3 PCR Error Rates as Measured by DGGE polymerase error rates. To maximize fi- Error rate per delity at the dNTP step (Fig. Polymerase nucleotide per cycle Reference 1A,B), any type of deoxynucleoside triphosphate pool imbalance should be T4 - 1/ 100,000 44 avoided. Similarly, stabilization of er- rors by polymerase extension from Modified T7 1/29,000 44 mispaired or misaligned primer- 1/23,000 45 termini (Fig. 1D) can be minimized by 1/18,000 46 reactions using short synthesis times, T. litoralis 1/42,000 45 low dNTP concentrations, and low en- 1/15,000 46 zyme concentrations. Additional im- Klenow 1/7,700 44 provements in fidelity can be made by further manipulating the reaction con- Taq 1/11,000 45 ditions. To perform high-fidelity PCR 1/5,000 46 with Taq polymerase, reactions should 1/4,800 44 contain a low MgC12 concentration,

22 PCR Methods and Applications Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

not in large excess over the total con- particular nucleotide sequence, since Riley, and A.F. Markham. 1988. centration of dNTP substrates, and be the phenomenon of "hot spots" for Diagnosis of ~l"antitrypsin deficiency buffered to -pH 6 (70~ using Bis-Tris polymerization errors has been clearly by enzymatic amplification of human Propane or PIPES (Table 2). These buff- established.(16) genomic DNA and direct ers have a pK a between pH 6 and pH 7 of polymerase chain reaction and a small temperature coefficient ACKNOWLEDGMENTS products. Nucleic Acids Res. 16: (ApKa/~ allowing the pH to be We are grateful to Drs. Theodora 8233-8243. maintained stably throughout the PCR Devereux and Neal Cariello for helpful 9. Vrieling, H., M.L. Van Roooijen, N.A. cycle. comments and critical evaluation of Groen, M.Z. Zdzienicka, J.W. Simons, For amplifications in which fidelity the manuscript. We would also like to P.H. Lohman, and A.A. Van Zeeland. is the critical issue, one should avoid thank Drs. Myron Goodman, William 1989. DNA strand specificity for UV- the concept that conditions generating Thilly, Lucy Ling, Pekka Mattila, and induced mutations in mammalian more DNA product are the better con- Neal Cariello for providing results prior cells. Mol. Cell. Biol. 9" 1277-1283. ditions. Reactions that optimize fidel- to publication. 10. Ennis, P.D., J. Zemmour, R.D. Salter, ity may be relatively inefficient for and P. Parham. 1990. Rapid cloning amplification. The total number of REFERENCES of HLA-A,B cDNA by using the poly- cycles should be kept to the minimum 1. Lacy, M.J., L.K. McNeil, M.E. Roth, merase chain reaction: Frequency and necessary to produce a feasible amount and D.M. Kranz. 1989. T-Cell receptor nature of errors produced in amplifi- of product DNA for the desired use. g-chain diversity in peripheral cation. Proc. Natl. Acad. Sci. 87: Thus, although using low pH, low lymphocytes. Proc. Natl. Acad. Sci. 86: 2833-2837. Mg 2+, and/or a low concentration of 1023-1026. 11. Loh, E.Y., J.F. Elliott, S. Cwirla, L.L. dNTPs may not produce an impressive 2. Frohman. M.A., M.K. Dush, and G.R. Lanier, and M.M. Davis. 1989. Poly- amount of amplification product as Martin. 1988. Rapid production of merase chain reaction with single- visualized on an ethidium bromide full-length cDNAs from rare sided specificity: Analysis of T-ceU stained-agarose gel, these conditions transcripts: Amplification using a receptor ~ chain. Science 243: may provide an adequate amount of single gene-specific 217-220. DNA for cloning purposes. Using a primer. Proc. Natl. Acad. Sci. 85: 12. Goodenow, M., T. Huet, W. Saurin, S. small number of cycles will also mini- 8998-9002. Kwok, J. Sninsky, and S. Wain- mize the contribution of mutations 3. Li. H., X. Cui, and N. Arnheim. 1990. Hobson. 1989. HIV-1 isolates are resulting from DNA damage at high Direct electrophoretic detection of rapidly evolving quasispecies: temperature. The DNA sequence of the allelic state of single DNA Evidence for viral mixtures and several independent clones from the molecules in human sperm by using preferred nucleotide substitutions. J. final PCR population should be deter- the polymerase chain reaction. Proc. Acquired Immune Defic. Syndr. 2: mined to verify that the original DNA Natl. Acad. Sci. 87: 4580-4584. 344-352. sequence has been maintained during 4. Jeffreys, A.J., R. Neumann, and V. 13. Eckert, K.A. and T.A. Kunkel. 1991. the amplification process. Ideally, the Wilson. 1990. Repeat unit sequence The fidelity of DNA polymerases used clones should be derived from separate variation in minisatellites: A novel in the PCR. In Polymerase chain PCR experiments to minimize the risk source of DNA polymorphism for reaction I: A practrical approach (eds. of overrepresentation of early polymer- studying variation and mutation by M.J. McPherson, P. Quirke, and G.R. use errors. single analysis. Cell 60: Taylor). IRL Press at Oxford Univer- The fidelity of DNA synthesis in 473-485. sity Press, Oxford. (In press.) vitro is limited by the least accurate 5. Ruano, G., K.K. Kidd, and J.C. 14. Reiss, J., M. Krawozak, M. Schloesser, reaction that occurs. For example, the Stephens. 1990. Haplotype of M. Wagner, and D.N. Cooper. 1990. T~ mispair is one of the most fre- multiple polymorphisms resolved by The effect of replication errors on the quently produced and most easily ex- enzymatic amplification of single mismatch analysis of PCR-amplified tended base substitution error.(35) Even DNA molecules. Proc. Natl. Acad. Sci. DNA. Nucleic Acids Res. 18" 973-978. under high-fidelity reaction condi- 87: 6296-6300. 15. Luria, S.E. and M. Delbruck. 1943. tions, most of the Taq polymerase er- 6. Dunning, A.M., P. Talmud, and S.E. Mutations of from rors were the result of T~ mispairs Humphries. 1988. Errors in the sensitivity to virus resistance. Genetics that led to A'T~G'C transitions.(13) polymerase chain reaction Nucleic 28" 491-511. Therefore, the upper limit for Taq poly- Acids Res. 16: 10393. 16. Kunkel, T.A. 1990. Misalignment- merase fidelity will be the frequency 7. Saiki, R.K., D.H. Gelfand, S. Stoffel, mediated DNA synthesis errors. with which the enzyme creates this S.J. Scharf, R. Higuchi, G.T. Horn, 29:8003-8011. mispair. In general, this limitation is K.B. Mullis, and H.A. Erlich. 1988. 17. Ripley, L.S. 1990. Frameshift not restricted to a particular mispair. Primer-directed enzymatic amplifica- mutation: Determinants of specifi- The sequence of the target DNA will tion of DNA with a thermostable city. Annu. Rev. Genet. 24: 189-213. have a profound influence on the gen- DNA polymerase. Science 239: 18. Streisinger, G., Y. Okada, J. Emrich, J. eration of mutations. Thus, the least 487-491. Newton, A. Tsugita, E. Terzaghi, and accurate reaction may be the result of 8. Newton, C.R., N. Kalsheker, A. M. Inoye. 1966. Frameshift mutations an exceptionally high error rate at a Graham, S. Powell, A. Gammack, J. and the . Cold Spring

PCR Methods and Applications 23 Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

Harbor Syrup. Quant. Biol. 31: 77-84. 32. Mendelman, L.V., J. Petruska, and immunodeficiency virus type 1 19. Bebenek, K. and T.A. Kunkel. 1990. M.F. Goodman. 1990. Base mispair model studies. Nucleic Acids Res. 18: Frameshift errors initiated by extension kinetics. Comparison of 999-1005. nucleotide misincorporation. Proc. DNA polymerase a and reverse tran- 44. Keohavong, P. and W.G. Thilly. 1989. Natl. Acad. Sci. 87: 4946-4950. scriptase. J. Biol. Chem. 265: Fidelity of DNA polyrnerases in DNA 20. Paabo, S., D.M. Irwin, and A.C. 2338-2346. amplification. Proc. Natl. Acad. Sci. Wilson. 1990. DNA damage promotes 33. Perrino, F.W. and L.A. Loeb. 1989. 86: 9253-9257. jumping between templates during Differential extension of 3' mispairs 45. Cariello, N.F., J.A. Swenberg, and T.R. enzymatic amplification. J. Biol. is a major contribution to the high Skopek. 1991. Fidelity of Thermo- Chem. 265: 4718-4721. fidelity of calf thymus DNA polymer- coccus litoralis DNA polymerase 21. Kuchta, R.D., P. Benkovic, and S.J. ase-a. J. Biol. Chem. 264: 2898-2905. (Vent) in PCR determined by Benkovic. 1988. Kinetic mechanism 34. Kunkel, T.A. 1988. Exonucleolytic denaturing gradient gel electro- whereby DNA polymerase I (Klenow) proofreading. Cell $3: 837-840. phoresis. Nucleic Acids Res. (in press). replicates DNA with high fidelity. 35. Bebenek, K., C.M. Joyce, M.P. 46. Ling, L.L., P. Keohavong, C. Dias, and Biochemistry 2 7:6716-6725. Fitzgerald, and T.A. Kunkel. 1990. W.G. Thilly. 1991. Optimization of 22. Goodman, M.F. 1988. DNA The fidelity of DNA synthesis the polymerase chain reaction with replication fidelity: Kinetics and catalyzed by derivatives of Escherichia regard to fidelity: Modified T7, Taq thermodynamics. Murat. Res. 200: coli DNA polymerase I. J. Biol. Chem. and Vent DNA polymerases. PCR 11-20. 265: 13878-13887. Methods and Applications (in press). 23. Loeb, L.A. and T.A. Kunkel. 1982. 36. Kunkel, T.A. and K. Bebenek. 1988. 47. Tabor, S. and C.C. Richardson. 1989. Fidelity of DNA synthesis. Annu. Rev. Recent studies of the fidelity of DNA Selective inactivation of the Biochem. 52: 429-457. synthesis. Biochim. Biophy. Acta 951: exonuclease activity of bacteriophage 24. Kunkel, T.A. 1985. The mutational 1-15. T7 DNA polymerase by in vitro specificity of DNA polymerase-[3 37. Eckert, K.A. and T.A. Kunkel. 1990. . J. Biol. Chem. 264: during in vitro DNA synthesis. J. Biol. High fidelity DNA synthesis by the 6447-6458. Chem. 260: 5787-5796. DNA polymer-ase. 48. Jeffreys, A.J., V. Wilson, R. Neumann, 25. Echols, H. and M.F. Goodman. 1991. Nucleic Acids Res. 18: 3739-3744. and J. Keyte. 1988. Amplification of Fidelity mechanisms in DNA 38. Lindahl, T. 1979. DNA glycosylases, human minisatellites by the polymer- replication. Annu. Rev. Biochem (in endonucleases for apurinic/apyrimi- ase chain reaction: Towards DNA press). dinic sites, and base-excision repair. fingerprinting of single cells. Nucleic 26. Mendelman, L.V., M.S. Boosalis, J. Prog. Nucleic Acid Res. Mol. Biol. 22: Acids Res. 16: 10953-10971. Petruska, and M.F. Goodman. 1989. 135-189. 49. Cariello, N.F., W.G. Thilly, J.A. Nearest neighbor influences on DNA 39. Loeb, L.A. and B.D. Preston. 1986. Swenberg, and T.R. Skopek. 1991. polymerase insertion fidelity. J. Biol. Mutagenesis by apurinic/apyrimi- Deletion mutagenesis during poly- Chem. 264:14415-14423. dinic sites. Annu. Rev. Genet. 20: merase chain reaction: Dependence 27. Preston, B.D., BJ. Poiesz, and L.A. 201-230. on DNA polymerase. Gene 99: (in Loeb. 1988. Fidelity of HIV-1 reverse 40. Paabo, S., R.G. Higuchi, and A.C. press). transcriptase. Science 242:1168-1171. Wilson. 1989. Ancient DNA and the 50. Tindall, K.R. and T.A. Kunkel. 1988. 28. Carroll, S.S., M. Cowart, and S.J. polymerase chain reaction. The Fidelity of DNA synthesis by the Benkovic. 1991. A mutant DNA poly- emerging field of molecular archae- Themms aquaticus DNA polymerase. merase I (Klenow fragment) with ology. J. Biol. Chem. 264: 9709-9712. Biochemistry 2 7:6008-6013. reduced fidelity. Biochemistry 30: 41. Newton, C.R., A. Graham, L.E. 51. Roberts, J.D., K. Bebenek, and T.A.; 804-813. Heptinstall, S.J. Powell, C. Summers, Kunkel. 1988. The accuracy of reverse 29. Kornberg, A. 1980. DNA replication. N. Kalsheker, J.C. Smith, and A.F. transcriptase from HIV-1. Science 242: W.H. Freeman and Company, San Markham. 1989. Analysis of any 1171-1173. Francisco, CA. point mutation in DNA. The amplifi- 52. Kunkel, T.A. and A. Soni. 1988. 30. Kunkel, T.A., R.A. Beckman, and L.A. cation refractory mutation system Exonucleolytic proofreading en- Loeb. 1986. On the fidelity of DNA (ARMS). Nucleic Acids Res. 17: hances the fidelity of DNA synthesis synthesis. Pyrophosphate-induced 2503-2516. by chick embryo DNA polymerase-,r misincorporation allows detection of 42. Wu, D.Y., L. Ugozzoli, B.K. Pal, and ]. Biol. Chem. 263: 4450-4459. two proofreading mechanisms. J. Biol. R.B. Wallace. 1989. Allele-specific Chem. 261: 13610-13616. enzymatic amplification of 13-globin 31. Doubleday, O.P., P.J. Lecomte, and genomic DNA for diagnosis of sickle M. Radman. 1983. A mechanism for cell anemia. Proc. Natl. Acad. Sci. 86: nucleotide selection associated with 2757-2760. the pyrophosphate exchange activity 43. Kwok, S., D.E. Kellogg, N. McKinney, of DNA polymerases. In Cellular D. Spasic, L. Goda, C. Levenson, and responses to DNA damage (eds. E.C. J.J. Sninsky. 1990. Effects of primer- Friedberg and B.A. Bridges), pp. template mismatches on the poly- 489-499. Alan R. Liss, New York. merase chain reaction: Human

24 PCR Methods and Applications Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press

DNA polymerase fidelity and the polymerase chain reaction.

K A Eckert and T A Kunkel

Genome Res. 1991 1: 17-24 Access the most recent version at doi:10.1101/gr.1.1.17

References This article cites 45 articles, 25 of which can be accessed free at: http://genome.cshlp.org/content/1/1/17.full.html#ref-list-1

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

Top View