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With Regard to Fidelity: and Vent Polymerases Lucy L Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Optimization of the Polymerase Chain Reaction with Regard to Fidelity: and Vent Polymerases Lucy L. Ling, Phouthone Keohavong, Cremilda Dias, and William G. Thilly Center for Environmental Health Sciences and Division of Toxicology, Whitaker College of Health Sciences and Technology, Massachusetts institute of Technology, Cambridge, Massachusetts 02139 The fidelity of DNA polymerases DNA amplification by the poly- Klenow fragment referred to in the used in the polymerase chain reac- merase chain reaction (PCR) O-3) can be original descriptions of DNA amplifica- tion (PCR) can be influenced by accomplished by DNA polymerases tion.(1,3,~~ However, the fidelity of Taq many factors in the reaction mix- from many sources.(1,2,4) Specific DNA has been reported to be 2 • 10 -4 er- ture. To maximize the fidelity of polymerases have been reported to ror/bp per duplication, (4,7-1~ which DNA polymerases in the PCR, pH, amplify with different efficiencies (i.e., renders it unsuitable for studies requir- concentrations of deoxynucleoside yield of sequence amplification per ing both low noise and high amplifica- triphosphates, and magnesium ion cycle) and fidelities (frequency of tion. Studies requiring a higher fidelity were varied. Denaturing gradient polymerase-induced errors), with the of amplification have used DNA poly- gel electrophoresis was used to kind and rate of error depending on merases such as modified T7 DNA separate the polymerase-induced the specific DNA polymerase and PCR polymerase (Sequenase) or T4 DNA mutants from wild-type DNA se- conditions. (4-9) Each error, once ini- polymerase,(4A3A4) which permitted quences. Thermolabile modified T7 tiated, is amplified along with the amplification with error rates of about DNA polymerase, therrnostable Taq, original wild-type sequences, increas- 3.4 x 10 -s and 0.3 x 10 -5 error/bp per and Vent DNA polymerases were ing the fraction of polymerase-induced duplication, respectively. (4) However, studied. Fidelity of all three DNA mutant sequences linearly with the both T4 and modified T7 DNA poly- polymerases was sensitive to con- number of amplification cycles. Under merases are thermolabile. centrations of deoxynucleoside low-fidelity conditions, this mutant Recently, a new thermostable DNA triphosphates, magnesium ion, and fraction can become significant: 40% polymerase called Vent DNA polymer- pH. Within conditions that permit- of the amplification products are ase was isolated and made commercial- ted efficient amplification, optimiza- mutant when a 200-bp fragment is ly available in a cloned form by New tion with regard to these three fac- amplified one million-fold by a DNA England Biolabs (Beverly, MA). Vent tors yielded an average error rate polymerase with an error rate of was reported to have a 3'-,5' in error/base pair incorporated of 10-4, (4) such as Klenow fragment.00) proofreading exonuclease. Os) Our 7.2 x 10 -s for Toq, 4.5 x 10 -s for For some purposes such as sequencing laboratory has been screening all avail- Vent, and 4.4 x 10 -s for modified T7 a wild-type gene, the polymerase- able thermostable polymerases and has (Sequenase) DNA polymerases. induced mutations are generally dis- found that this new thermostable DNA tributed over the sequence of interest polymerase, under conditions de- so that an accurate consensus sequence scribed for primer extension, permitted is usually obtained.O 1,12) For studies of amplification with a useful degree of fi- mutants within wild-type populations, delity. Reaction conditions such as pH, it is vital that the polymerase-induced concentrations of dNTP, and mag- mutant sequences do not mask the nesium ion can greatly affect the fidel- preexisting mutant sequences.Ol,13A4) ity of DNA polymerases.(S,7,9,16-20) Eck- Due to its thermostabiUty, Taq ert and Kunkel used the M13mp2 fidel- DNA polymerase has been the most ity assay and found that conditions of widely used DNA polymerase. It does" low pH with low and equimolar con- not have to be added every cycle, as centrations of dNTP to Mg 2+ increased was necessary for the thermolabile the fidelity of Taq.(7,9) We have ex- 1:63-699 byCold Spring Harbor Laboratory Press ISSN 1054-9803/91 $3.00 PCR Methods end Applications 63 Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press tended their observations using gel to ensure a homogeneous popula- PCR reaction mixture was removed denaturing gradient gel electrophoresis tion of the desired fragment of human and electrophoresed on 6% polyacryl- (DGGE) to separate and enumerate all HPRT exon 3 and flanking intron 2. amide gel together with a series of polymerase-induced mutant sequences This template was amplified from 10 s authentic standards whose numbers of from the correctly amplified sequences. copies to about 1012 copies and molecules are known. The gel was To maximize the fidelity of Taq, Vent, polyacrylamide gel purified. An aliquot stained with ethidium bromide and and Sequenase, varied concentrations (101~ copies) was further amplified the PCR products of the correct size of dNTPs, pH, and magnesium were lO0-fold, under identical conditions were measured by comparing the yield tested iteratively, and the optimal con- with primer P4 end-labeled by T4 poly- of the ultraviolet-induced fluorescence ditions with regard to these variables nucleotide kinase.(13) The number of emitted by the ethidium bromide have been defined. We have confirmed DNA copies generated was measured at molecules intercalated into the DNA of the finding of Eckert and Kunkel(7, 9) different cycles to permit calculation of the samples of DNA of the standard. and defined conditions in which the efficiency. Conditions that maintained The error of this method of quantita- Taq polymerase operates with a three- exponential increases in DNA per cycle tion is about twofold. However, this fold improvement in fidelity during were used throughout this work. The would affect the efficiency only slight- amplification in PCR relative to the 32p-end-labeled DNA of the correct size ly. For instance, a calculated efficiency most widely used reaction condi- was then purified from polyacrylamide of 70% due to 107-fold amplification tions. (4) More importantly, we find gel, boiled, and reannealed so that after 30 PCR cycles, would be 75% if that the thermostable Vent polymerase each strand of any PCR-generated the DNA was actually twice as much. under optimal conditions yielded a mutant homoduplexes could be hy- The efficiency of amplification was cal- PCR amplification with both high fi- bridized to the complementary strand culated from the formula: delity and high efficiency which of the excess wild-type products to Final DNA copy number should be of general use to researchers form heteroduplexes. These hetero- (1 + y)n = using PCR. duplexes were then separated from the Initial DNA copy number wild-type homoduplexes by denatur- MATERIALS AND METHODS ing gradient gel electrophoresis. For where Y is the efficiency per cycle and Materials each PCR condition, the total hetero- n is the number of PCR cycles under Taq polymerase was obtained from duplex and the correctly amplified conditions of exponential ampliflca- Perkin Elmer Cetus (Norwalk, CT). homoduplex fractions were measured tion.(1,2,14) Vent polymerase, purified from for the calculation of fidelity in er- Thermococcus litoralis, and its recom- ror/base pair incorporated per duplica- Denaturing Gradient Gel binant form cloned in Escherichia coli tion. Electrophoresis (DGGE) (designated Rec-Vent in this paper) To separate DNA duplexes containing were obtained from New England PCR Procedure only homologous wild-type sequences Biolabs (Beverly, MA). Sequenase pH, dNTP, and Mg 2§ concentrations (WT homoduplexes) from the mutant/ (modified T7 DNA polymerase) was ob- were experimental variables. MgSO 4 wild-type heteroduplexes, denaturing tained from United States Biochemicals was used for Taq and Vent, while gradient gel electrophoresis was (Cleveland, OH). 2'-deoxynucleoside MgC12 was used for Sequenase. For employed. (4) DNA was dissolved in a 5'-triphosphates (dNTPs) were ob- Vent and Taq, the PCR reaction mix- 30-~1 solution of 400 mM NaC1, 10 mM tained as 100 mM solution from ture was 10 mM KC1, 10 mM Tris buffer at pH 7.5, 2 mM EDTA, Pharmacia (Piscataway, NJ). Primer P1, (NH4)2SO 4, 0.1% Triton X-100, 0.1 boiled for 5 min, and reannealed for 5 5 ' -CATATATTAAATATACTCAC-3 '; ~g/~l BSA, 1 ~M each primer and 20 hr at 65~ The DNA was ethanol primer P2, 5'-TCCTGATITTA3TFC- mM Tris buffer. Four units of enzyme precipitated and separated on a TGTA-3 '; and primer P4, 5 ' -GACTGA- were added for every 100 ~l of reaction denaturing gradient gel. The gel con- ACGTCTTGCTCGAG-3' were obtain- volume, with 3 more units of Taq poly- sists of a 12.5% polyacrylamide gel ed from Synthetic Genetics (San Diego, merase added after 30 cycles. The (acrylamide/bis-acrylamide -- 37.5:1), CA). [y-32p]ATP (6000 Ci/mmole) was amplification cycle consisted of 94~ containing a linearly increasing gradi- from New England Nuclear (Boston, for 1 rain, 53~ for 2 min, and 70~ ent of denaturant, parallel to the direc- MA) and T4 polynucleotide kinase was for 2 min. For Sequenase, the reaction tion of electrophoresis, of 19-29% from New England Biolabs (Beverly, mixture was 3 pM each primer, and 10 (vol/vol) where 100% denaturant is 7 M MA). The reagents in the 5' DNA mM Tris buffer. The amplification cycle urea/40% formamide.(21) The gel was terminus labeling system were from consisted of boiling for 1 min, cooling run for 15 hr at 150 volts while sub- Bethesda Research Lab (Gaithersburg, down to 37~ (1 minute), adding 1 merged in TAE buffer (40 mM Tris/20 MD).
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