Table 11.4 Selected Properties of Common Thermostable DNA Polymerases Heat Stability Proofreading Processivity Extension 5′-3′ vity NTP/ Rate(dNTP/ 四gDNA Absent Present9at975°C 60-150 (40-60at concentration Stoffel fragment Absent21at975°C 5-10 Tth dna Trace Present 30.40 Absent DN Absent50at97.5°C Present Absent1140at95°C 102 Absent Absent1140at95°C 1 form) Present absent 1380at95°Cc es gB- (aka Deep 480at100°C Present bsent402at95°C aka vent l08at100° Herculase P Present anced Tbr DNa Absent Present150at96°C lamer Platinum Pr/ P Absent 72 100-20 100-300 Platinum Tag Present 6at95°c 5060 60-150 Absent40at95°C Abse 30at75°C Mth pol Absent12at75°C ThermalAc Present Absent 5-fold greater than Polymerase Hot Tub(T. Present Absent Similar to Taq data from Perler, Kumar, and Kong (1996). Data provided by H Hogrefe, Stratagene, Inc bs catalog. 2000 Z Kelman (JBC 274: 28751): present according to Perler. Data provided by D. titus, MJ Re es Inc Data provided by J Ambroziak, Clonetech Laboratories Inc. sawyer et al.(1993). PCR Methods application pp275-286 ta provided by Amersham Pharmacia Biotech, Ine PCR 30
PCR 301 Table 11.4 Selected Properties of Common Thermostable DNA Polymerases Heat Stability Proofreading (min before Processivity Extension (3¢–5¢ 5¢–3¢ 50% activity (dNTP/ Rate (dNTP/ Enzyme exonuclease) Exonuclease remains) binding) s/mol) Taq DNA Absent Present 9 at 97.5°C 50–60 60–150 polymerase (40–60 at 95°C. depending on protein concentrationa,b Stoffel fragment Absent Absent 21 at 97.5°C 5–10 130 Tth DNA Absent Present 25 polymerase rTth XL Trace Present 30–40 AmpliTaq CS Absent Absent 50–60 UlTma DNA Present Absent 50 at 97.5°C polymerase (low) Pfu DNA Present Absent 1140 at 95°Ca 10a 60 polymerase (native and recombinant) Pfu DNA Absent Absent 1140 at 95°Ca 11a polymerase (exo-form) (Pyrococcus Present Absent 1380 at 95°Cc >80 species GBD) (aka Deep 480 at 100°C Vent®) Tli Pol Present Absent 402 at 95°Cc 7 67 (aka Vent®) 108 at 100°C Herculase Present Presenta enhanced DNA polymerase Tbr DNA Absent Present 150 at 96°C polymerase (DynazymeTM) e Platinum Pfxf Present Absent 720 at 95°C 100–200 100–300 180 at 100°C Platinum Taqf Absent Present 96 at 95°C 50–60 60–150 Advantaq Absent Absent 40 at 95°C 40 Polymeraseg Tac Pol Present Absent 30 at 75°C Mth Pol Present Absentd 12 at 75°C ThermalAceTM Present Absent 5-fold greater Pyolobus than Taq fumariush DNA Polymerase Hot Tub (T. Present Absent Similar to Taq flaius)i Source: Unless otherwise noted, all data from Perler, Kumar, and Kong (1996). aData provided by H. Hogrefe, Stratagene, Inc. bNew England Biolabs Catalog, 2000. c Z. Kelman (JBC 274 : 28751); present according to Perler. dData provided by D. Titus, MJ Resesarch, Inc. e Data provide by D. Hoekzema, Life Technologies Inc. f Data provided by J. Ambroziak, Clonetech Laboratories Inc. gData provided by Invitrogen, Inc. hLawyer et al. (1993). PCR Methods & application pp. 275–286. i Data provided by Amersham Pharmacia Biotech, Inc
Fidelity Fidelity could be defined as an enzyme's ability to insert the roper nucleotide and eliminate those entered in error. As thor oughly reviewed by Kunkel (1992), fidelity is not a simple matter there are several steps di the polymeri of dna whe cussions of fidelity focus on the proofreading function provided exonuclease activity. Cline, Braman, ogrefe(1996)compared the ermostable DNA polymerases side by side, taking care to optimize the con- ditions for each enzyme. They observed the following fidelity rates mutation frequency/bp/duplication), in order: Pfu(1.3 x10-)> Deep vent(27×100)>vent(28×10)>q(8.0×10-)>exo Pfu and UITma(-5 x 10-. These and similar data should be viewed in relative rather than absolute terms, because assay methods affect the absolute number of detected misincorpora- tions(Andre et al., 1997), and sample source can affect the performance of enzymes differentially and unpredictably(Abu Al-Soud and Radstrom, 1998) Proofreading activity can also reduce PCR yield, especially in reactions that generate long PCR products. The greater time required to extend the fragment increases the chance of primer degradation by the 3-5 exonuclease activity(de Noronha and Mullins, 1992 and Skerra, 1992). The problem of reduced yield can be corrected by including an enzyme with strong proofreading activity into a PCR reaction with a polymerase that lacks a strong proofreading activity(Barnes, 1994; Cline, Braman, and Hogrefe, 1996: MJ Research Inc Application Bulletin, 2000) Is a higher reaction temperature always helpful and necessary? No. For most DNA-based PCR. the consensus is that hot-start PCR increases both sensitivity and yield by preventing nonspe cific PCR product formation(Faloona et aL., 1990). Higher tem- eratures can melt secondary structures, but there are limitations to the use of heat. Very high denaturation temperatures can also damage DNA, through depurination and subsequent fragmenta tion, especially during long PCR reactions( Cheng et al., 1994).It can also increase hydrolysis of RNA in one step RT-PCR in the presence of magnesium ions(Brown, 1974). In order to reduce heat-induced damage, incorporation of additives such as DMSO is used(see later section on additives Choosing an enzyme with specialized activities will not produ 302 Aoyagi
Fidelity Fidelity could be defined as an enzyme’s ability to insert the proper nucleotide and eliminate those entered in error. As thoroughly reviewed by Kunkel (1992), fidelity is not a simple matter; there are several steps during the polymerization of DNA where mistakes can be made and corrected. Still most practical discussions of fidelity focus on the proofreading function provided by an enzyme’s 3¢–5¢ exonuclease activity. Cline, Braman, and Hogrefe (1996) compared the fidelity of several thermostable DNA polymerases side by side, taking care to optimize the conditions for each enzyme.They observed the following fidelity rates (mutation frequency/bp/duplication), in order: Pfu (1.3 ¥ 10-6 ) > Deep Vent (2.7 ¥ 10-6 ) > Vent (2.8 ¥ 10-6 ) > Taq (8.0 ¥ 10-6 ) > exoPfu and UlTma (~5 ¥ 10-5 ). These and similar data should be viewed in relative rather than absolute terms, because assay methods affect the absolute number of detected misincorporations (André et al., 1997), and sample source can affect the performance of enzymes differentially and unpredictably (Abu Al-Soud and Radstrom, 1998). Proofreading activity can also reduce PCR yield, especially in reactions that generate long PCR products. The greater time required to extend the fragment increases the chance of primer degradation by the 3¢–5¢ exonuclease activity (de Noronha and Mullins, 1992 and Skerra, 1992). The problem of reduced yield can be corrected by including an enzyme with strong proofreading activity into a PCR reaction with a polymerase that lacks a strong proofreading activity (Barnes, 1994; Cline, Braman, and Hogrefe, 1996; MJ Research Inc. Application Bulletin, 2000). Heat Stability Is a higher reaction temperature always helpful and necessary? No. For most DNA-based PCR, the consensus is that hot-start PCR increases both sensitivity and yield by preventing nonspecific PCR product formation (Faloona et al., 1990). Higher temperatures can melt secondary structures, but there are limitations to the use of heat. Very high denaturation temperatures can also damage DNA, through depurination and subsequent fragmentation, especially during long PCR reactions (Cheng et al., 1994). It can also increase hydrolysis of RNA in one step RT-PCR in the presence of magnesium ions (Brown, 1974). In order to reduce heat-induced damage, incorporation of additives such as DMSO is used (see later section on additives). Choosing an enzyme with specialized activities will not produce 302 Aoyagi
the desired results unless the appropriate conditions are applied For example, UITmaM DNA polymerase has a pH optimum for olymerase activity of 8.3 and exonuclease activity at pH 9.3 Bost et aL., 1994). Likewise, presence of metal ions can favor one activ- ity over the other for many polymerases. Long pcr Additives such as single-stranded binding protein(Rapley, 1994), T4 gene 32 protein(Schwarz, Hansen-Hagge, and Bartram, 1990), and proprietary commercial products may increase the production efficiency of long PCR. However, fidelity was also e cruc al to the replication of large products via PCR (Barnes, 1994). By supplementing PCR reactions containing Taq dna polymerase(which lacks proofreading activity) with proofreading-rich Dna polymerase, Barnes generated fragments up to 35 kb. Bear in mind that proofreading activity can potentially reduce yield, especially with large PCr products As discussed above, this problem can be avoided by utilizing a combination of polymerases that possess and lack strong proof- reading activity The availability of specialized, designer enzymes are an attrac- tive strategy that shouldnt be ignored. However, selecting the right enzyme(s)is one step among many, and cant guarantee the desired result. One near-term example is the importance of enzyme concentration. The concentration of polymerase applied to a PCr reaction ranges from one to four units per 100uL Greater concentrations can increase formation of nonspecific PCR products. The importance of optimizing other parameters such as buffer component, primer design, and cycling conditions is shown in Table 11.3 and Table 11.5 How Can Nucleotides and Primers Affect a Pcr Reaction? Nucleotide concentration The standard concentration of each nucleotide in the final reac- ion is approximately 200 uM, which is sufficient to synthesize 12. 5 ug of DNA when half of the dNTPs are incorporated. Adding more nucleotide is unnecessary and detrimental. Too much nucleotide reduces specificity by increasing the error rate of the polymerase and also chelates magnesium, changing the optimal magnesium concentration(Gelfand, 1989; Coen, 1995) Primer concentration The standard primer concentration is 100 to 900nM; too much primer can increase the formation of nonspecific products. It is PCR 303
the desired results unless the appropriate conditions are applied. For example, UlTmaTM DNA polymerase has a pH optimum for polymerase activity of 8.3 and exonuclease activity at pH 9.3 (Bost et al., 1994). Likewise, presence of metal ions can favor one activity over the other for many polymerases. Long PCR Additives such as single-stranded binding protein (Rapley, 1994), T4 gene 32 protein (Schwarz, Hansen-Hagge, and Bartram, 1990), and proprietary commercial products may increase the production efficiency of long PCR. However, fidelity was also shown to be crucial to the replication of large products via PCR (Barnes, 1994). By supplementing PCR reactions containing Taq DNA polymerase (which lacks proofreading activity) with proofreading-rich Pfu DNA polymerase, Barnes generated fragments up to 35kb. Bear in mind that proofreading activity can potentially reduce yield, especially with large PCR products. As discussed above, this problem can be avoided by utilizing a combination of polymerases that possess and lack strong proofreading activity. The availability of specialized, designer enzymes are an attractive strategy that shouldn’t be ignored. However, selecting the right enzyme(s) is one step among many, and can’t guarantee the desired result. One near-term example is the importance of enzyme concentration. The concentration of polymerase applied to a PCR reaction ranges from one to four units per 100mL. Greater concentrations can increase formation of nonspecific PCR products. The importance of optimizing other parameters, such as buffer component, primer design, and cycling conditions is shown in Table 11.3 and Table 11.5. How Can Nucleotides and Primers Affect a PCR Reaction? Nucleotide Concentration The standard concentration of each nucleotide in the final reaction is approximately 200mM, which is sufficient to synthesize 12.5mg of DNA when half of the dNTPs are incorporated. Adding more nucleotide is unnecessary and detrimental. Too much nucleotide reduces specificity by increasing the error rate of the polymerase and also chelates magnesium, changing the effective optimal magnesium concentration (Gelfand, 1989; Coen, 1995). Primer Concentration The standard primer concentration is 100 to 900 nM; too much primer can increase the formation of nonspecific products. It is PCR 303