A REVIEW ARTICLE

MICHIGAN STATE THERMUS AQUATICUS DNA POLYMERASE

UNIVERSITY

Taq DNA Polymerase | Maison, D. 2009

Maison, D 2009 Taq DNA Polymerase

TAQ DNA POLYMERASE: THE CORE OF ENZYMATIC DNA AMPLIFICATION BY POLYMERASE CHAIN REACTION

DAVID P. MAISON

MICHIGAN STATE UNIVERSITY LYMAN BRIGGS COLLEGE, MICHIGAN STATE UNIVERSITY CYSTIC FIBROSIS RESEARCH LABORATORY

Abbreviated Title: Taq DNA Polymerase

Corresponding Author: Douglas B. Luckie, Ph.D., Michigan State University Department of Physiology

Editor/Reviewing Authors: Richard B. Maison, MPA., Robert B. Miller College – School of Business Michael J. Ramar, Michigan State University Lyman Briggs College

Word Count*: 5,035

Last Date of Revision: 24/09/2009 KEYWORDS

• Amplicon – a term for any small, replicating DNA fragment • Processivity – the number of nucleosides added by the enzyme before it detaches • Polymerization rate – nucleotides/second • Fidelity – an assessment of the precision of a replication • Thermophile – an organism with a required growth temperature between 45°C and 80°C • Cofactor – inorganic complement of an enzyme reaction, usually a metal ion • Coenzyme – a small molecule essential for the activity of some enzymes; helps an enzyme catalyze a particular reaction by binding with it • CATH – (Class, Architecture, Topology, Homologous Superfamily) – classification of protein structures • SCOP – Structural Classification Of Proteins • PFAM – The Protein Family Database Classification • Chelate - of or noting a heterocyclic compound having a central metallic ion attached by covalent bonds to two or more nonmetallic atoms in the same molecule.

Abstract

Though the polymerase chain reaction is one of the most widely used microbiological techniques today, its advancement and optimization since its use became universal in 1984 has been slow or non-existent. The foremost rationale for immense expansion of PCR is the use of a thermostable enzyme in the reaction, eliminating the need for additional introduction of enzyme after each cycle. The optimization and enhancing protocols that are commonly found at present in many scientific journals have to do primarily with altering annealing temperatures, changing cycle time, or doing numerous reactions to find the most favourable conditions for a specific reaction. Though these can be decent techniques once an amplicon has been replicated to finish, they are not universal. The aim of this paper is to initially describe the fundamentals of Taq DNA polymerase, its structure, as well as the activity, and then conclusively to examine its use in the Polymerase Chain Reaction.

* Word Count is of all pages, including Title Page, Abstract, References, Figures and Tables

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ENZYME HISTORY & INTRODUCTION

History While the credit for the use of Taq DNA Polymerase in the polymerase chain reaction is given to Kary R. Mullis for his idea on using a thermostable polymerase1, there are several key researchers whose discoveries made this possible. One who instigating this in 1955 with the discovery of the enzyme known as a DNA polymerase1, was Arthur R. Kornberg. Kornberg, along with his colleagues at Stanford University, revealed that the Taq enzyme was capable of extending an oligonucleotide primer by continuously polymerizing the addition of an additional nucleotide at the 3’ end, corresponding to the template sequence. From there, they established that the solution required for this DNA polymerase enzyme required building blocks in the form of nucleotide triphosphates2. Following the publication of his discoveries, Kornberg, along with Severo Ochoa from New York University College of Medicine, was awarded the Nobel Prize in Physiology or Medicine 1959 “for their discovery of the mechanisms in the biological synthesis on ribonucleic acid and deoxyribonucleic acid.”3 In all probability, the most significant researcher in terms of where in vitro DNA amplification is at present is Thomas D. Brock. In the middle of the 1960’s, Brock began to research at Yellowstone National Park in Montana. Though thermophilic bacteria were not believed to exist at the time and bacteria as a family were not thought to live above the environmental temperature of 80°C, Brock nonetheless studied the growth in hot springs and hydrothermal vents.4 His most significant discovery during his exploration was that of the bacteria Thermus aquaticus in 1965, which was the first of the archaebacteria to be encountered.5 His isolation of the polymerase within this bacterium paved the way for DNA amplification in biotechnology. With his publication of ‘Life at High Temperatures: Evolutionary, Ecological, and Biochemical Significance of Organisms Living in Hot Spring is Discussed’ in the November 1967 issue of Science, Brock spawned the thermophilic field of microbiology.6 Despite the fact that was not credited with the discovery of any of the factors of enzymatic DNA amplification, it is without question that without Kary R. Mullis, research would not be where it is today. In a sum of his own words, Mullis describes his idea to use a thermostable DNA polymerase rather than a polymerase that is degraded after ever cycle, as a straightforward DNA sequencing idea that he perfected on U.S. Highway 101 while on his way to Mendocino County with a friend. Numerous ideas passed through his mind but he couldn’t remove the image of two primers being replicated, with 3’ ends en route for crossing paths yet on complementary strands. From that night forward he began to refine and continuously study his brilliant. The emergence of the polymerase chain reaction using thermostable DNA polymerase occurred in 1984 while Mullis was at the Cetus Corporation. Mullis was awarded the Nobel Prize in Chemistry 1993 for his creation of 2 the polymerase chain reaction. FIGURE 1 - KARY R. MULLIS - FROM NOBEL PRIZE DATABASE

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The patents for Taq DNA polymerase and the polymerase chain reaction were ultimately

purchased from the Cetus Corporation by Hoffmann-La Roche for $330 million. Thermus aquaticus & Taq DNA polymerase The bacterium Thermus aquaticus, as previously stated, was discovered in the thermal hot springs (Lower Geyser Basin) of Yellowstone National Park in Montana by Thomas Dale Brock. The bacteria is known as a thermophile since the optimal temperature for the organisms growth lies within the temperatures of 45°C and 80°C.7,10 The scientific classification of the phylum is a group of bacteria known as the Deinococcus-Thermus, which are comprised of cocci bacterium that are known for their defiant character to environmental danger.8 However, as interesting and amazing as this bacteria is, it is what lies within that is of concern to biotechnology – Thermus aquaticus DNA Polymerase.

The DNA polymerase that resides within the Thermus aquaticus bacterium is a type of enzyme non-existent in mammals; it is one that is thermostable. Due to the origin of this enzyme in hot springs, the most advantageous temperatures lie in a range between 70-75°C.9 Part of the polymerase A family, Taq is a paralog to other prokaryotic polymerases such as DNA polymerase I and T7 DNA polymerase as well as eukaryotic polymerases γ and θ.14,15,16 Researchers at Yale University have, in point of fact, determined that the amino acids in the polymerase domain of Taq DNA polymerase are 51% identical to that of DNA Polymerase I.17 Similar characteristics and properties have been found between Taq DNA polymerase and the DNA polymerase I of E. coli; primarily in the exonuclease and dNTP binding sites. This relationship pertains to the use of recombinant Taq polymerase in the polymerase chain reaction (see Significance in PCR). The Taq enzyme is categorized into several families by the CATH, SCOP and PFAM classification systems based primarily upon domains but also on several other various factors. The CATH classification system catalogues based upon class, architecture, topology, and homologous superfamily (Table 1).11 The remaining approach, known as the SCOP classification, classifies the enzyme by class, fold, superfamily, family, domain, and finally by species (Table 3).13 The PFAM, protein families database, classification system segregates the enzyme based upon chain, then classifies by description and type (Table 2).12 Taq polymerase was awarded the Molecule of the Year award by Science in 1989.10

TABLE 1A - CATH CLASSIFICATION11 Species Domain Class Fold Superfamily Family

Thermus 5’ to 3’ SAM domain- C-terminal C-terminal exonuclease α-proteins aquaticus domain of Taq like subdomain subdomain Thermus 5’ to 3’ PIN domain- Catalytic exonuclease α/β PIN domain-like aquaticus domain of Taq like domain Exonuclease Thermus domain of Ribonuclease Ribonuclease H- DnaQ-like 3’- prokaryotic α/β aquaticus DNA H-like motif like 5’ exonuclease polymerase

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TABLE 2 - SCOP CLASSIFICATION13 Domain Class Architecture Topology Homology 1taqA01 α/β 3-layer aba sandwich Rossman fold 5’-nuclease 1taqA02 α Orthogonal bundle Domain 1 C-terminal subdomain Nucleotidyltransferase, 1taqA03 α/β 2-layer sandwich domain 5 Taq polymerase, Chain 1taqA04 α Up-down bundle Chain T, domain 4 T, domain 4 1taqA05 α/β 2-layer sandwich α-β Plaits 1taqA06 α Orthogonal bundle Domain 1 C-terminal subdomain

TABLE 3 - PFAM CLASSIFICATION12 PFAM Chain PFAM ID Description Type Clan ID Accession DNA polymerase A PF00476 DNA_pol_A Family N/A family A Taq polymerase, A PF09281 Taq-exonuc Domain N/A exonuclease 5/’-3/’ A PF01367 5_3_exonuc exonuclease, C- Domain N/A terminal SAM fold 5/’-3/’ exonuxlease, N- A PF02739 5_3_exonuc_N Domain N/A terminal resolvase- like domain

ARCHITECTURE & CATALYTIC ACTIVITY

Structure at the Active Site The structure of Taq DNA polymerase involves three domains (Figure 2) that combine in the polymerization act of the enzyme. The domains, independently, are responsible for individual acts that ultimately result in the replication of template DNA. The polymerase domain, known as the “thumb” of the enzyme, is used to bind incoming dsDNA. The “palm of the enzyme is the 3’ exonuclease domain; this is where catalytic and polymerization activity occurs, as well as where the divalent cations (primarily Mg2+) are bound and in a state of flux with aspartic acid residues. The activity of the exonuclease domain has a requisite for a bound metallic divalent cation. The third and final domain of Taq is the 5’ nuclease domain, which will interact with ssDNA as well as bind the incoming nucleotides between the hydrophobic pocket created by Tyr526 and Asp 480.14 (For more information on deoxynucleotide triphosphate binding, see section “The Sequencer.”) Amino acid residues 1-290 comprise 5’ nuclease activity as well as the amino terminus.

FIGURE 2 - DOMAINS OF TAQ DNA POLYMERASE. From top, the polymerase domain which binds the incoming double-stranded dna; the 3’ exonuclease domain, which is responsible for the catalytic activity of the enzyme as well as the harborage site from the divalent matalic cations; and the 5’nuclease domain, which regulates incoming deoxynucleotide triphosphates between Tyr526 and Asp480.

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FIGURE 3 - CARTOON REPRESENTATION OF THERMUS AQUATICUS DNA POLYMERASE WITH LABELED STRUCTURES IN THE "HAND" ANALOGY.27 The polymerase family of enzymes is commonly interpreted in terms of a “hand,” as described in the Structure of the Active Site section. The nucleotide binding site is referred to as the fingers, the palm is the 3’ exonuclease domain, and the thumb is the polymerase domain when DNA is bound. Original image from reference 14.

Open & Closed forms of Enzyme & Active Site The active site of DNA polymerase is where the reaction catalyzed by the enzyme will occur. The inner lining of the active site of Taq DNA polymerase involves the use of three aspartic acid residues that interact with cations (or a molecule that possesses the highest binding affinity) through hydrogen bonding interactions. This allows the cations to then stabilize both ssDNA and deoxynucleotide triphosphate which leads to the condensation reaction of 5’ nucleophilic attack by DNA phosphate on the 3’ hydroxyl group of nucleotide (Figure 4). The distribution between open (Figure 5) and closed (Figure 6) conformations occurs by means of binding substrates. In the instance of Taq, the open conformation would be present until the act of polymerization commences. The entirety of the enzyme itself folds in a flux between open and closed formations when substrates are bound and not bound, respectively. This in turn galvanizes the active site, the crux of the enzyme, into a more condensed center. The compression of the active site allows the catalytic divalent cation to move into one of the two binding sites, allowing the divalent cation pre-chelated to a deoxynucleotide triphosphate to enter the second active site. The now bound cations are able to stabilize the ssDNA and dNTP allowing 5’ to 3’ polymerization to occur. (For more information please see section “When Push Comes to Shove.”)

H H H H H H H O N NH O N NH O N NH H H HN N

O N O HN N O N N N N N N N N N N N N N N

O O O O O O H H H H H H H H H H H H FIGURE 4 - DIVALENT CATION STABILIZATION FOR 3’ OXYANION HO O HO O HO O HO O HO O - O P P P P P OH NUCLEOPHILIC ATTACK ON 5’ α-PHOSPHATE. The stabilization by O O O O O O O O O O the catalytic Mg901 (green right) and nucleotide bound Mg902 (green left) O ions on the negatively charged triphosphate backbone of the N NH deoxynucleotide phosphate allows for nucleophilic attack by the 3’ O O O - N O P O P O P O N NH2 oxyanion. The products released by this reaction include magnesium - - - O O O O chelated pyrophosphate and water (from condensation). The aspartic acid

OH residues within the Rossman folds are Asp610 and Asp785. Image generated on ACD/ChemSketch. (Not shown: H2O molecules also interact HO O with Mg901 during this phase) H N 2

O 2+ 2+ OH Mg Mg

OH O NH O 2 HO H N OH 2 O O HO

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a) b)

FIGURE 5 - OPEN CONFORMATION OF THERMUS AQUATICUS DNA POLYMERASE. In this phase, the polymerase only has a single divalent cation bound, shown as Mg901 (green). This cation is known as the catalytic cation and is an unconditional necessity for Taq DNA polymerase to become active. If denied this cation, the enzyme is prone to degradation and will not engage in catalytic (polymerization) activity. a) The enzyme, as a whole, in the open conformation. b) The active site of the enzyme in the 3’ exonuclease domain. PDB ID 2ktq.

a) b)

FIGURE 6 – CLOSED (ACTIVE) CONFORMATION OF THERMUS AQUATICUS DNA POLYMERASE. Contrary to the open conformation displayed in Figure 3, the closed/active conformation is the configuration present during catalytic activity. a) The entirety of the enzyme displayed in active configuration. b) The active site of the Taq with both the Mg901 (catalytic) and Mg902 (nucleotide bound) metallic divalent cations shown. PDB ID 3ktq.

ENZYME CHARACTERISTICS

Processivity, Error Rate, & Accuracy Routinely, it has been assessed that Taq polymerase is capable of polymerizing amplicons up to a length of 4,000 base pairs.9 While this is not considered optimal for polymerizing entire proteins, it is ideal for use in the polymerase chain reaction for the majority of cases. This number is also an approximate determination of what the actual processivity of Taq is. In actuality, it is the accessibility of the sequential nucleotide that is the most important determinant in the rate at which Taq is able to incorporate nucleotides onto the developing amplicon,18 assuming that the divalent concentration in the environment is optimal so as to eliminate competition and reduce excessive stabilization of the DNA backbone.

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The error rate of the enzyme conveys the number of wrongly incorporated nucleotides onto the template ssDNA per total nucleotides added. This can be expressed in two ways. Within terms of a single base pair, Taq possesses an error rate for base substitution between 1.1×10!! and 8.9×10!! errors per base pair added19 (averaged at 1/9000).20 Also, in respect to frame shift errors, which are exponentially more drastic. Taq polymerase’s frame shift error frequency is ~ 1 frame shift per 40,000 base pairs added.2 There are various equations to determine to error rate of an enzyme through experimental testing (Table 4). Though these numbers are minute and do not seem drastic, if an error occurs at the beginning of a polymerase chain reaction, that error may be incorporated into the millions of sequential copies that will use the initial amplicons as templates.9

The accuracy of the enzyme is essentially the inverse of the error rate and expresses the number of nucleotides incorporated correctly before the event of a misincorporation occurs.9 The ! equation for accuracy is thus, �������� = !, where “E” denotes the prior determined error rate. The approximant accuracy of Taq is between 9,091 and 11,236 base pairs.

TABLE 4 - ERROR RATE EQUATIONS Equation Source EF = error frequency mf = mutation fraction MFo=obs mutation freq following replication

MFb=the background mutation frequency �� ��! − ��! �! fo=the freq of expression of newly �� = synthesized strand 20 �! Nd=the # of nucleotides within the 250- nucleotide target region known to yield a mutant phenotype with a specific mutational

change. (base substitution. – Nd=110; frame shift – Nd=160) 1 �� = ER = error rate 18 �������� ����� ���� ������ �� ����� 2 1 �� = ER = error rate 21 �������� �������� ���� 0.6 # ���������� ����� E = error rate % ��������� d = overall # of amplimers present in final � = 9 �×���×100 del = amplicon length *E ~ 1.8×10!!

Fidelity The fidelity of an enzyme is an overall assessment on the exactness of the amplimer product.9 This cannot generally be calculated by an equation before polymerization occurs, but rather must be factored on the reactants within the reaction. This can be assessed if the enzyme has been provided optimal concentrations; for Taq this is equivalent to the accuracy discussed above.

Polymerization Rate When reactions are carried out in an in vitro environment, it is necessary to determine the rate at which the product will generated. The most advantageous environment as well as environment must be determined for proper catalysis to occur. This is with the intention that the enzyme will not break away from the template DNA strand before the amplimer is completely polymerized.

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FASTA Sequence of Thermus aquaticus DNA Polymerase GenBank: BAA06775.1 >gi|507891|dbj|BAA06775.1| DNA Polymerase [Thermus aquaticus]

MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAK A PSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILT ADKDLYQLLSDRIHALHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEE WGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGS LLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARG LLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRL EGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQL ERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRL HTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIR VFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSF PKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKL FP RLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE

LOCUS BAA06775 832 aa linear BCT 26-JAN-2008 DEFINITION DNA Polymerase [Thermus aquaticus]. ACCESSION BAA06775 VERSION BAA06775.1 GI:507891 DBSOURCE locus TTHDNAP accession D32013.1 KEYWORDS . SOURCE Thermus aquaticus ORGANISM Thermus aquaticus Bacteria; Deinococcus-Thermus; Deinococci; Thermales; Thermaceae; Thermus. REFERENCE 1 AUTHORS Ishino,Y., Ueno,T., Miyagi,M., Uemori,T., Imamura,M., Tsunasawa,S. and Kato,I. TITLE Overproduction of Thermus aquaticus DNA polymerase and its structural analysis by ion-spray mass spectrometry JOURNAL J. Biochem. 116 (5), 1019-1024 (1994) PUBMED 7896728 REFERENCE 2 (residues 1 to 832) AUTHORS Ishino,Y., Ueno,T., Miyagi,M., Uemori,T., Imamura,M., Tsunasawa,S. and Kato,I. TITLE Direct Submission JOURNAL Submitted (30-JUN-1994) Contact:Yoshizumi Ishino Laboratory of Protein Chemistry and Engineering, Dept. of Genetic Resources Technology, Faculty of Agriculture, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University; 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka 812-8581, Japan

SIGNIFICANCE IN THE POLYMERASE CHAIN REACTION

When one visualizes the amplification of DNA by means of the polymerase chain reaction, we focus upon the deoxyribonucleic acid amplicon. However, as the cells in our body depend on the concentrations of surrounding ions, each salt ion in the solution plays a crucial role in PCR and ultimately the continued activation of the polymerase. To ensure that the optimal activity of the Thermus aquaticus DNA polymerase is continuous throughout the polymerization process, each substrate and species within the reaction environment must be accounted for and its effect noted.

Additive Effects

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The use of additives in the polymerase chain reaction has become a widespread phenomenon across the biotechnological community. This section is a brief introduction to the primary additives used in the polymerase chain reaction.

BETAINE O The molecular compound of Betaine ((trimethylammonio)acetate) is a widely used enhancer for polymerizing amplicons that are rich is GC content. The ideal concentration of Betaine varies with the size of the total reaction C 22 mixture. For a 30µl reaction, the ideal volume is 0.8M ; whereas the final H concentration is generally employed between 1.0-1.7M for a 50µl reaction.23 NH2

DMSO H3C Dimethyl sulfoxide is a compound that is often tested with the aforementioned S O Betaine due to their similarity in terms of usage, as well as the restraining effects that is generally present between the two. Also used for the H3C amplification of GC rich templates, the expression of DMSO and Betaine simultaneously is noted to greatly reduce the overall product yield. The final concentration that is often used for dimethyl sulfoxide is between 2-10%22. However, at higher concentrations this additive has been shown to inhibit the Taq polymerase enzyme by as much as 50%.23

FORMAMIDE

More ideal than the previously mentioned additives in terms of effect on Taq DNA O polymerase, is the additive formamide. Although it does not inhibit the activity of the enzyme, it is a more troublesome additive in that the concentration must be C optimized. Generally formamide is used at 1.5-2.0% with an inhibitory effect at H 23 NH greater than 10%. Although this additive is considered somewhat new and 2 advanced, its narrow window of effectiveness makes it undesirable in many reactions.24

HO DTT SH Dithiothreitol is a reagent commonly used in redox reactions and generally known as Cleland’s reagent. Although few studies have been conducted to HS study the effect of DTT independently, it has shown to increase yield when OH used in combination with other additives such as betaine and dimethyl sulfoxide. The principle reaction concentration is in the range of 0.8mM to 3.2mM.22

BOVINE SERUM ALBUMIN Bovine Serum Albumin, commonly denoted BSA, is one of the most common additives used at present. It is particularly useful in amplifying ancient DNA templates but is also known to stabilize enzyme as well as reduce the effect of contaminants that may possess inhibitory effects.22,23

TMAC CH 3 Tetramethylammonium chloride is an additive that would be optimal in mutation + CH N 3 testing or in a sizeable amplification. TMAC has been shown to reduce, if not H3C - 28 Cl eliminate, non-specific priming. The final reaction concentration for this H3C additive varies between 15mM and 100mM. *All images generated using ACD/ChemSketch

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Magnesium Concentration Potentially the most important component in terms of reaction optimization for Taq DNA polymerase is the concentration of free magnesium ions available in solution. The term “free” is used due to the reality that there are many other components and substrates within a reaction that compete for these ions. Components including DNA and primers for use in stabilization, dNTPs that chelate these ions for use in polymerization, and water than forms an ionic barrier around the magnesium. As discussed in Structure of the Active Site, the activity of the polymerase depends on magnesium ions and without these it will not function. If the magnesium concentration is too low and quickly depleted, this will result in a low product yield as the optimal amount of enzymes are not being employed to amplify. However, lower concentrations of magnesium are generally preferred when the fidelity of the DNA synthesis is critical.25 If the magnesium concentration is too high, the DNA and oligonucleotides will be hyper-stabilized and amplification will not occur. For these reasons, it is currently necessary to optimize the concentration of this divalent cation for optimal yield of amplicon.

Concentration of Taq DNA polymerase & Unit Definition In respect to the previously discussed magnesium concentration, the concentration of enzyme used must also be ideal for several reasons. The primary being that the Taq readily competes for magnesium ions, thus reducing the amount in the overall reaction for other components that require the ion (deoxynucleotide-triphosphates). If the concentration of enzyme is too low, it would be expected to obtain to low product yield as there are few enzymes available to amplify. This could also result in a high magnesium concentration due to the lack of chelation by the active site. A high concentration could result in non-specific priming and primer-dimers; since the enzymes function is to polymerize, it will do just that with whatever template and substrate are available.

The quantity of Taq is generally expressed in terms of units per µl(U). A single unit consists of the amount of enzyme that catalyzes the incorporation of 10nmol of nucleotides into acid precipitable material in 30 minutes between 70 and 74°C.9,19,26,29,30 The number of units used per reaction depends on several factors of the reaction and must be optimized accordingly, but is generally between 0.25 and 2.5U per 50µl reaction.

Half-Life & Temperatures As stated in the introduction to Thermus aquaticus DNA polymerase, Taq exhibits optimal activity within the temperature range of 70-75°C. Although important this is not the principal reason that temperature that needs to be focused upon in a PCR. The half-life of the enzyme is the most important aspect when determining the length and number of cycles that can be ran for a particular reaction.

The half-life of the enzyme is a measure of the time required for one half of the enzymes of a given amount to denature. If a higher temperature is more consistent than the half-life will decrease at a coinciding pace. For example, even though the half-life of the enzyme is approximately 40 minutes at 95°C, this number reduces to 5 minutes when the temperature is increased only 2.5°C to 97.5°C. Theoretically, this means that in a reaction with 25 cycles with a denaturation temperature of 95°C for 40 seconds, roughly 50% of the enzyme will have denatured by the completion of the cycles.9

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Optimum Reaction Temperature for Thermus aquaticus DNA Polymerase

180 160 140 120 100 80 60

pMOLEs DNTPs Incorporated 40 20 0 0 20 40 60 80 100 Temperature (°C)

FIGURE 7-OPTIMUM REACTION TEMPERATURE FOR THERMUS AQUATICUS DNA POLYMERASE *Data obtained from reference 31

160 140 120 100 80 60 Half-life (minutes) 40 20 0 92 93 94 95 96 97 98 Temperature (°C)

FIGURE 8 - HALF-LIFE OF THERMUS AQUATICUS DNA POLYMERASE2,12,13

Recombinant forms of Taq & Other Thermostable Polymerases Since Taq is generally not the most ideal enzyme due to its limited processivity and lack of 3’-5’ proofreading, there have been many recombinant forms of the enzyme commercially made to enhance and optimize the overall performance. These recombinant forms are most commonly expressed as vectors in E. coli and created to contain modifications suited to particular functions. The following table gives an outline of many alternative forms of the enzyme available today.

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TABLE 5 - COMMERCIALLY AVAILABLE THERMOSTABLE DNA POLYMERASES Thermostability 5’-3’ 3’-5’ Polymerization Enzyme pH Optimum (minutes @95°C) Proofreading Proofreading Rate (nuc/s) AmpliTaq 40 Yes No 75 7-7.5 Stoffel 80 No No >50 - Ampliterm - No No - - Pyra >300 No No - 7-7.5 TTH94 20 Yes No >33 7-7.5 Tfl 40 Yes No 16 7-7.5 Tfu 300 No Yes 64 - DeepVent 400 No Yes >80 - Vent 1380 No Yes - - Tli 400 No Yes 33 7-7.5 Proofstart >240 No Yes - - PFU92 >120 No Yes 60 8-9 Pfx - No Yes - - Pwo - - Yes - - ULTma >50 No - - - ThermalAce >4 - Yes - - *Table obtained from reference 9

ACKNOWLEDGEMENTS

SPECIAL THANKS TO THE FOLLOWING: Douglas B. Luckie – for his continuous support and generous funding of Taq23PC

Jon Stoltzfus, Ph.D –for his collaboration and consultation in the function of enzymes Michael Haenisch, M.Sc. –for his critique and encouragement

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Maison, D 2009 Taq DNA Polymerase

REFERENCES

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Maison, D 2009 Taq DNA Polymerase

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