Taq DNA Polymerase Paper Wo Experiments1

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 2 Maison, D 2009 Taq DNA Polymerase 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 3 Maison, D 2009 Taq DNA Polymerase 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 4 Maison, D 2009 Taq DNA Polymerase 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

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