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CHAPTER 1.0 : INTRODUCTION
Polymerase Chain Reaction (PCR) is widely used in biotechnology and molecular biology for various studies including genetic research and molecular studies. It is a technique developed by Kary Mullis where he was thinking about his DNA sequence experiment before this technique came into his mind (Mullis 1990). Soon, various techniques such as qPCR, RT-PCR, nested PCR and so on were being introduced. PCR is used in amplifying desire DNA templates to obtain certain amount of DNA in order to carry out deeper studies. However, to run PCR effectively, heat stable DNA polymerases are required to replicate DNA templates, ordinary DNA polymerases would denature if heated to high temperature and required to be replenished in each PCR cycle. There are many heat stable DNA polymerase isolated from thermophilic archaea bacteria, and the most commonly used ones are Taq, Pfu and Vent as well as others modified polymerase such as Klentaq. These enzymes have their very own fidelity in replicating DNA templates in which the frequency of mismatch base pairing varies in each enzymes (Cline, Braman et al. 1996).
Because of the natural habitats of these thermophilic archaeal bacteria are almost impossible to mimic in laboratories, there are difficulties in obtaining heat stable DNA polymerases from these thermophilic bacteria. Most scientists choose to buy these polymerases from companies as they are being commercialized. However, thanks to recombinant DNA technology, genes of heat stable polymerases could be obtained and cloned into plasmids, and expression could be done to obtain large amount of these DNA polymerases, by using bacterial system, such as most commonly studied bacteria E. coli. This allows production of readily use DNA polymerases available in laboratory, though commercially available DNA polymerases are produced the same way by companies.
Although heat stable DNA polymerases are readily produced in laboratory, effectiveness in amplifying DNA templates is not achieved in some commonly use DNA polymerases. Taq polymerase, extracted from thermophilic archaeal bacteria Thermus aquaticus is one of the most widely use heat stable DNA polymerase due to its significant fidelity. However, it lacks 3’ ïƒ 5’ exonuclease activity (Tindall and Kunkel 1988) and this is the main factor which contributes to high error rate of mismatch base pairing during DNA amplification. High error rate of DNA amplification has to be avoided to prevent alteration of products, leading to inaccurate results. Hence, Taq polymerase is not suitable in amplifying long template, which frequency of mismatch base incorporation is much higher (Cline, Braman et al. 1996).
Pfu polymerase, isolated from hyperthermophilic archaeal bacteria Pyrococcus furiosus, is a proofreading polymerase, capable of 3’ ïƒ 5’ exonuclease activity. The ability of proofreading of Pfu makes it a higher fidelity polymerase than Taq (Lundberg, Shoemaker et al. 1991), which means it induces much lesser error during DNA amplification by excising and replacing mismatched base pairs. However, it has a low processivity of approximately 0.84, in which it dissociates from DNA template easily (Wang, Prosen et al. 2004). This creates a problem in duplicating long DNA templates.
In order to obtain an ultimate DNA polymerase with proofreading ability and high fidelity, Sso7d domain is fused to C-terminus of Pfu polymerase (Wang, Prosen et al. 2004). Sso7d, a heat stable DNA binding protein, isolated from Sulfolobus solfataricus, has a property that binds to non-specifically double strand DNA (dsDNA) (Baumann, Knapp et al. 1994). This property would allow the Pfu polymerase to gain processivity by binding to primer-template DNA complex with higher affinity and thus a higher fidelity.
- Research Objectives
- To fuse Sso7d domain to Pfu to increase its fidelity and processivity
- To express, extract and purify Taq and Pfu-fusion polymerases
- To run assays to determine the fidelity and processivity of the polymerases by PCR
CHAPTER 2.0 : LITERATURE REVIEW
2.1 The Archaea Domain
The discovery of the Archaea organisms has changed the view of scientists on the world of biology that organisms are found in extreme conditions such as hot springs, environments with high salt content, acid and base surroundings. The world of biology is then classified into three main domains, the Bacteria, Archaea and Eukarya based on ribosomal RNA (rRNA) sequences (Woese and Fox 1977). The domain Archaea is further divided into two main phyla, namely Euryachaeota and Crenarchaeota (Garrity and Holt 2001). Then, phyla of Korarchaeota (Barns, Delwiche et al. 1996) and Nanoarchaeota (Huber, Hohn et al. 2002) are being proposed to fit in newly discovered species that are quite distinctive from the phyla Euryachaeota and Crenarchaeota. In 2008, a new phylum, Thaumarchaeota (Brochier-Armanet, Boussau et al. 2008) is proposed. It is clearly that there are still many archaeons and other organisms to be discovered and classified.
Thermophiles, can be referred as organisms which grow optimally at thermophilic condition of temperature between 65oC to 85oC, and hyperthemophiles which grow optimally at temperature 85oC to 110oC. The discovery of thermophilic and hyperthermophilic has brought great interests to biologists on questioning the organization and structure of various proteins and enzyme under such extreme conditions, especially its DNA polymerases. Because of the thermostability of these thermophilic DNA polymerases, they are utilized by biologists in various studies such as the polymerase chain reaction (PCR) (Saiki, Gelfand et al. 1988).
2.2 The Polymerase Chain Reaction (PCR)
It was a Friday night where Kary Mullis was driving to Mendocino Country with his asleep chemist friend. He was thinking about his DNA sequencing experiment where four different types of radioactive dideoxynucleotide triphosphates (ddNTPs) are prepared in four tubes, each tube contains different type of radioactive ddNTPs. Then, DNA templates, DNA primer and DNA polymerase are added to allow complementary base pairing in order to determine the base sequence of the DNA template. Because of the ddNTPs, there is no hydroxyl group (OH–) at the 3’ carbon, the DNA polymerase could not extend further due to the absence of 3’ oxygen end. Hence, the polymerase reaction will stop and different sizes of oligonucleotides are obtained, and they can be separated by electrophoresis and hence many bands could be observed to determine the bases complementary to the radioactive ddNTPs.
However, it is a rather slow process yet accuracy is not promising. Hence, Mullis was thinking that it could be more definitive and result in a more convincing outcome, by using two primers, or oligonucleotides with different sizes, instead of one. This allows Mullis to bracket targeted base pairs to be identified, and base sequences of both complementary strands could be read. In his experiments, there was always the presence of stray traces of nucleotides which could be extended before terminated by ddNTPs. Soon, Mullis realized that he could not add any phosphatase to cleave away any unwanted nucleotide, because the phosphatase would also cleave his ddNTPs. Hence, he added polymerase to deplete the nucleotides into the extending oligonucleotides before adding ddNTPs. By increasing the temperature, the oligonucleotides would separate from the DNA target and unextended primers could pair to DNA target by chance after cooling the mixture and hence ddNTPs could bind.
Then, Mullis realized that, what if the oligonucleotide has been completely extended instead of one or two bases, and the extended DNA could be complementary to the target DNA, in other words, a complete DNA is synthesis, complementary to the target DNA! Suddenly, a looping process of mathematical programme came into his mind, that, if the process of extension oligonucleotides were to loop, where heating, cooling, adding polymerase and nucleotides are repeated, a fixed length of DNA fragment would be generated, and he could deduce the looping process leads to exponential growth of the quantity of the targeted DNA, and this is where he invented the polymerase chain reaction! Mullis has been working for several months to optimize and improve his PCR method, where pH of the buffer, salt contents (Mg2+), size of DNA templates, temperature of mixture buffer, time spent in each stage of PCR with different temperature and number of cycles. Klenow fragment was first used for PCR. Because Klenow fragment is extracted from E. coli, the enzyme is not heat stable and it has to be replenished every cycle. Until the discovery of heat stable polymerase from Thermus aquaticus named Taq. The use of the thermophilic DNA polymerase was a major breakthrough, because depletion of DNA polymerase is no longer happening, and they are not need to be added at each cycle to replenish the denatured polymerase (Mullis 1990).
2.3 Heat Stable DNA Polymerases and Proteins
2.3.1 Taq DNA Polymerase
Thermus aquaticus, a thermophilic archaeon bacteria which is isolated from hot springs produces a single polypeptide, heat-stable DNA polymerase named Taq (or Taq Pol I), which derives directly from the name of the archaeal bacteria. Thermus aquaticus. T. aquaticus grows optimally at temperature of 65oC to 85oC with optimum temperature for its DNA synthesis activity at 80oC (Chien, Edgar et al. 1976). Taq polymerase becomes popular in laboratories to carry out PCR due to its thermostability and hence enzyme repletion of each cycle of PCR is not required. Studies have shown that the molecular weight of Taq polymerase is approximately 63kDa, which is relatively small compared to DNA polymerases isolated from E. coli and it requires magnesium ions (Mg2+) for optimum polymerize activity (Chien, Edgar et al. 1976). Taq polymerase has strong 5’ ïƒ 3’ DNA catalytic activity but it lacks 3’ ïƒ 5’ exonuclease activity or proofreading activity (Tindall and Kunkel 1988). Absence of proofreading can be overcame by adding small amount of DNA polymerase with proofreading ability such as the Klenow fragment from DNA polymerase I of E. coli.
188.8.131.52 Characteristics of Taq Polymerase in PCR
Fidelity of Taq polymerase is considered to be adequate to amplify DNA of sufficient quantity for various studies under any in vitro synthesis condition, given that various parameters such as the concentration of deoxynucleotide triphosphate (dNTP) and magnesium chloride (MgCl2), and pH of buffer are in optimum conditions for in vitro synthesis and amplification of DNA. Studies have shown that under conditions of equimolar concentration of dNTPs and MgCl2, the mutation rate of base substitution and frameshift of nucleotides can be as low as 1×10-5 and 1×10-6, respectively (Eckert and Kunkel 1990). Taq shows an average primer extension of 22 nucleotides with processivity of 0.95 (Wang, Prosen et al. 2004). This indicates Taq could incorporate quite a number of nucleotides before it is dissociated from the primer-template DNA complex.
2.3.2 Pfu Polymerase
Pfu polymerase, a hyperthermophilic DNA polymerase purified from hyperthermophilic archaeal bacteria named Pyrococcus furiosus, which grows optimally at 100oC (Uemori, Ishino et al. 1993). This hyperthermophile isolated mainly from shallow submarines and geothermal environment found in deep sea utilizes fermentation-like metabolism rather than reduction of So. The molecular weight of Pfu is found to be about 90kDa, estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Uemori, Ishino et al. 1993). Pfu polymerase could be considered as the most efficient polymerase used in amplifying DNA samples in vitro due to its error correcting property of 3’ ïƒ 5’ exonuclease activity, where mismatched base pairs are excised and replaced. It is shown that the mutation rate induced by Pfu polymerase is 7~10 times lower than that of Taq polymerase (Barnes 1994). Hence, Pfu polymerase has a higher fidelity than non-proofreading Taq polymerase, which lacks the 3’ ïƒ 5’ exonuclease activity (Lundberg, Shoemaker et al. 1991).
184.108.40.206 Characteristics of Pfu Polymerase in PCR
Error rate induced by Pfu polymerase when it is used in amplifying DNA samples in vitro is found to be significantly low. This is true when several conditions and parameters are under specific circumstances. Under optimum conditions, such as 200µM of each dNTP and 2mM MgSO4, in buffer with pH of 8.8, the error rate of Pfu polymerase is 1.3×10-6 (Cline, Braman et al. 1996). Study shown that the average primer extension rate of Pfu is about 6 nucleotides with processivity of approximately 0.84 (Wang, Prosen et al. 2004). This indicates that processivity of Pfu is quite low, making it not suitable in amplifying long target DNA.
2.3.3 Sso7d Protein Domain
Sso7d, thermoacidophilic DNA binding protein, isolated from Sulfolobus solfataricus, which grows optimally at 80oC in pH range of 2~4 (Zillig, Stetter et al. 1980). It is a small, basic, 7,150Da (Edmondson and Shriver 2001), thermophilic, and it is found in abundance by binding non-specifically to DNA, preferably dsDNA (Baumann, Knapp et al. 1994). This also helps in stabilizing the genomic DNA. It is believed that it resembles eukaryotic histone proteins. In fact there are several DNA binding proteins from Sulfolobus solfataricus, only one form of Sso7 has been characterized, it is known as Sso7d which is studied base on the homology of Sac7d (Choli, Henning et al. 1988). Moreover, Sso7d is found to perform several functions including induce negative supercoiling (Lopez-Garcia, Knapp et al. 1998), aids in annealing of complementary DNA strands (Guagliardi, Napoli et al. 1997) and chaperon activity of renaturation and disassembaly of protein aggregates in ATP hydrolysis -dependent manner (Guagliardi, Cerchia et al. 2000).
2.3.4 The Fusion Protein
The fusion protein, Pfu-S, or commercially known as Phusion, is actually a Pfu polymerase, with Sso7d fused to its C-terminus (Wang, Prosen et al. 2004). Sso7d has a property that binds to non-specifically dsDNA, more specifically the primer-template DNA, which enables the Pfu polymerase to grip and catalyze polymerization of nucleotides to the ssDNA with higher efficiency. This in turns, leads to high fidelity rate of the Pfu-S protein as the fusion protein gains processivity. Study shown that the Pfu-S protein demonstrates a processivity of value 0.98 and rate of average primer extension of approximately 55 nucleotides. Sso7d acts as a polymerase enhancer, hence it does not affect the fidelity and processivity of the protein. The Sso7d has a great heat stability and its size is small (Knapp, Karshikoff et al. 1996), hence it has no effects on the thermostability of the fusion protein. Pfu-S remains thermostable as its original form of Pfu polymerase with approximately 10 hours of half-life at 97.5oC. Study has shown that that templates up to 15kbp can be amplified with Pfu-Sso7d fusion proteins in PCR reaction (Wang, Prosen et al. 2004).
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