Polymerase Chain Reaction (PCR) is a process that uses primers to amplify specific cloned or genomic DNA sequences with the help of a very unique enzyme. PCR, an acronym for Polymerase Chain Reaction, allowed the production of large quantities of a specific DNA from a complex DNA template in a simple enzymatic reaction. PCR has transformed the way that almost all studies requiring the manipulation of DNA fragments may be performed as a results of its simplicity and usefulness [1]

 

In the 1980s, Kary Mullis and a team of researchers at Cetus Corporation conceived of a way to start and stop a polymerase's action at specific points along a single strand of DNA. Mullis also realized that by harnessing this component of molecular reproduction technology, the target DNA could be exponentially amplified. [1]

 

This DNA amplification procedure was based on an in vitro rather than an in vivo process. Cell-free DNA amplification by PCR was able to simplify many of the standard procedures for cloning, analyzing, and modifying nucleic acids. Previous techniques for isolating a specific piece of DNA relied on gene cloning – a tedious and slow procedure. PCR, on the other hand Kerry Mullis stated "lets you pick the piece of DNA you’re interested in and have as much of it as you want”. When other Cetus scientists eventually succeeded in making the polymerase chain reaction perform as desired in a reliable fashion, they had an immensely powerful technique for providing essentially unlimited quantities of the precise genetic material molecular biologists and others required for their work. Since the first report in1985, more than 5000 scientific papers were published by 1992. [1]

 

PCR was thought to be conceived by Dr. Kerry Mullis in 1983 while working at the Cetus Corporation in Emeryville, CA. However, some pioneering work was also done by Gobind Khorana in 1971 who described a basic principle of replicating a piece of DNA using two primers. Progress then was limited by primer synthesis and polymerase purification issues. In Mullis’s head, the invention grew from a theoretical scheme to perform limited dideoxynucleotide sequencing of unique human genes using synthetic oligonucleotides for the purpose of diagnosing common human disease mutations. An obvious obstacle to such a direct sequencing strategy was the high complexity of the human genome (3.3 X 10 power 9 base pairs). Thus, a second oligonucleotide or primer was added to block the progression of the synthesis of the first primer. Later however, this second primer was included to bind to the other DNA strand, so that each strand of the mutant allele would contribute to the eventual signal. If the scheme involving simultaneous hybridization of primers to each strand was modified by heating the mixture and then repeating the annealing and extension steps, then the primary signal would be increased even further. Repeating the steps would enable the products of the first round to be duplicated in the second cycle, to yield two copies. Repeating the cycle again would result in four copies, et cetera. Several weeks passed before this great idea was attempted. Two primers were synthesized to be perfectly complementary to each end of the 110 base pair region of a cloned segment of the human b-globin gene, the amplification was performed, and the products were identified by acrylamide gel electrophoresis. The end result was the anticipated 110 base pair DNA fragment and the beginning of PCR as a basic technique in molecular biology. [1]

 

In Mullis's original PCR process, the enzyme was used in vitro (in a controlled environment outside an organism). The double-stranded DNA was separated into two single strands by heating it to 96°C. At this temperature, however, the E.Coli DNA polymerase was destroyed so that the enzyme had to be replenished after the heating stage of each cycle. Mullis's original PCR process was very inefficient since it required a great deal of time, vast amounts of DNA-Polymerase, and continual attention throughout the PCR process. The inactivation of the Klenow fragment of Escherichia coli DNA polymerase I at the high temperature required for strand separation required the addition of enzyme after the denaturation step of each cycle. Prior to 1988, anyone conducting a PCR reaction procedure was obliged to sit patiently by a series of water baths or heating blocks and add a fresh aliquot of E.Coli DNA polymerase after each denaturation step, which was typically carried out by immersing the reaction vessel in boiling water for ½ a minute to 3 minutes. This rather tedious step was eliminated by the introduction of a thermostable DNA polymerase, the Taq DNA polymerase once, at the beginning of the PCR reaction. The thermostable properties of the DNA polymerase activity were isolated from Thermus aquaticus (Taq) that grow in geysers of over 110C, and have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction. The Taq enzyme can withstand repeated heating to 94C and so each time the mixture is cooled to allow the oligonucleotide primers to bind the catalyst for the extension is already present. However, higher annealing temperatures were not established until the single "most important development of PCR development”, the purification and commercial distribution of a heat-resistant DNA polymerase from the thermophilic bacterium Thermus aquaticus (Taq). [1]

 

Examination of the PCR amplification mechanism reveal its simplicity but also its elegance . Oligonucleotide primers are first designed to be complementary to the ends of the sequence to be amplified, and then mixed in molar excess with the DNA template and deoxyribonucleotides in an appropriate buffer. Following heating to denature the original strands and cooling to promote primer annealing, the oligonucleotides each bind to a different strand of the target fragment. The primers are positioned so that when each is extended by the action of a DNA polymerase, the newly synthesized strands will overlap the binding site of the opposite oligonucleotide. As the process of denaturation, annealing, and polymerase extension is continued the primers repeatedly bind to both the original DNA template and complementary sites in the newly synthesized strands and are extended to produce new copies of DNA. The end result is an exponential increase in the total number of DNA fragments that include the sequences between the PCR primers, which are finally represented at a theoretical abundance of 2n, where n is the number of cycles. [1]

 

In addition to the production of double-stranded, blunt-ended DNA fragments which may be formed by PCR, two other features of the PCR scheme contribute greatly to the utility of PCR. First, the position of binding of the primers defines the boundaries of the amplified fragment and therefore the prior molecular cloning requirement of restriction endonuclease recognition sites is not required for PCR. As only a limited number of DNA sequences are restriction sites, PCR greatly increases the flexibility of choice of fragment size and composition. Secondly, it is not necessary for PCR oligonucleotides to be exactly complementary to the template DNA. "Tails” may be added to the 5’ end of the primer to introduce sequences within the priming sites which thus may be exploited to introduce restriction endonuclease recognition sites or other useful sequences such as mutations into the amplified DNA. This phenomena allowed the emergence of PCR as a method for rapid DNA cloning. [1]

 

Molecular cloning has benefited from the emergence of PCR as a technique. Direct cloning was first conducted using a 110 bp DNA fragment amplified by PCR and oligonucleotide primers which contained restriction endonuclease recognition sites added to their 5’ ends. These sites were used to facilitate cloning of the amplified DNA into an M13 plasmid (17). The 110 bp fragment was also sequenced to confirm that this approach was a rapid yet reliable approach to cloning. [1]

 

Despite its huge popularity, PCR has certain limitations as a method for selectively cloning specific DNA sequences. In order to construct specific oligonucleotide primers that permit selective amplification of a particular DNA sequence, some prior sequence information is usually necessary. This normally means that the DNA region of interest has been partly characterized previously, often following prior cell-based DNA cloning. However, a variety of approaches have been developed that reduce or even exclude the need for prior DNA sequence information concerning the target DNA. Previously uncharacterized DNA sequences can sometimes be cloned using PCR with degenerate oligonucleotides if they are members of a gene or repetitive DNA family at least one of whose members has previously been characterized. In some cases, PCR can be used effectively without any prior sequence information concerning the target DNA to permit indiscriminateamplification of DNA sequences from a source of DNA that is present in extemely limited quantities. Therefore, although PCR can be applied to ensure whole genome amplification, it does not have the advantage of cell-based DNA cloning in offering a way of separating the individual DNA clones comprising a genomic DNA library.

 

The amount of PCR product obtained in a single reaction is also much more limited than the amount that can be obtained using cell-based cloning where scale-up of the volumes of cell cultures is possible. The efficiency of a PCR reaction will vary from template to template and according to various factors that are required to optimize the reaction but typically only comparatively small amounts of product are achieved.
Although the theoretical yield of PCR is exponential, the actual yield of a PCR is much less indicating that the scheme is operating with less than its maximum potential. For example, the amount of product at each cycle eventually levels off. This plateau may be explained by the following phenomena. First, some of the template may never be available due to strand breaks or failure of the DNA to dissociated from other macromolecules during purification and the initial thermocycles. Secondly, the amount of enzyme is finite and eventually activity may decrease. Thirdly, as the concentration of the double-stranded product reaches high levels, competition increases between annealing of template (PCR product) to primer and reannealing of the complementary template strands.

 

An obvious and many times great disadvantage of PCR as a DNA cloning method has been the size range of the DNA sequences that can be cloned. Unlike cell-based DNA cloning where the size of cloned DNA sequences can approach 2 Mb, reported DNA sequences cloned by PCR have typically been in the 0.1 5 kb size range, often at the lower end of this scale. Small fragments of DNA can usually be amplified easily by PCR, however it becomes increasingly more difficult to obtain efficient amplification as the desired product length increases. Barnes recognized a target length limitation to PCR amplification of DNA. He used a combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent) to conduct high fidelity long PCR. At least 35 kb of bacteriophage lambda can be amplified to high yields from 1 ng of lambda DNA template. Use of this method yielded increased base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment. Other conditions have been identified for effective amplification of longer targets, including amplification of up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA. The conditions for these long PCRs included increased pH, addition of glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and the use of a secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity. The "long PCR" protocol maintained the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics. Generally, the conditions for long range PCR involve a combination of modifications to standard conditions with a two-polymerase system. This provides optimal levels of DNA polymerase and 3’to 5’ exonuclease activity which serves as a proofreading mechanism. [1]

 

As PCR became more widely used, scientists rapidly learned more about it and, as a result, learned that the PCR had its strong points and its deficiencies. Very quickly, PCR demonstrated its power to amplify very small amounts (e.g., a single copy) of template nucleic acid and to amplify different nucleic acids (e.g., DNA and RNA). At the same time, laboratory personnel learned that this biochemical reaction had a unique deficiency; namely, a strong susceptibility to contamination from its own product. Early experience with the PCR soon showed that additional precautions were needed. Part1.1 of the pre clinical trials of the Biotech tropicana, Inc SMARThivPACK diagnostic platform aims to review the basic requirements of PCR laboratory whose operations will give reliable and contamination free results. [2]

 

 

Design of a PCR facility may be considered at two levels, 1) Whether a new laboratory is to be built or, 2) whether a PCR laboratory will be adapted to an existing infrastructure.

 

In building a new PCR infrastructure, basic requirement of a chemistry laboratory should be considered. The infrastructures should be built in accordance with recommended general, ventilation, electrical, and plumbing standards, for a chemical laboratory. The basic requirements are described in the University of Hawaii II, guidelines for a chemical laboratory. [3]

 

Whether a PCR laboratory is being set in a new building or is being adapted to an existing building, the basic PCR specific requirements must be met. In settings up a PCR laboratory, we recommend the guidelines set forth in Lo et al. 1988 [4]; Kwok and Higuchi 1989 [5]; and Mifflin T [2].

 

In designing a PCR laboratory, contamination avoidance is the key factor that is considered. PCR protocols are typically executed in a linear fashion, sample preparation, PCR reaction assembly, PCR execution, and post-PCR analysis. These four basic steps are grouped into two major steps, a pre-PCR step and a post-PCR step. PCR laboratories are designed accordingly. A typical conventional PCR facility is a two rooms laboratory, one for pre-PCR activities and the second for post-PCR activities. There are application specific variants of the typical two rooms laboratory. These variants will be discussed in part 1.2 of the Biotech tropicana, Inc PCR laboratory publication series, for the resource-poor settings. The typical two rooms PCR laboratory organization is described by Mufflin from the University from Virginia, USA, in reference 2, page 8. [2]. In this work we simulate the two rooms model as described by Mufflin using card board for specific materials. Our card board model permits us to re-construe and vizualise our reconstruction at a lower cost than conventional method. Available at http://btitechtrials.ucoz.com/photo.

 

In addition to knowledge of applicable scientific principles, vizualisation may elucidate potential difficulties that may arise in a pratical "real” application. The card board model is cheap and simple, and of valid scientific quality. Resource and expertise are scarce in the developing world. The data derived from our card boar models are scientifically valid, since the models are built from objective scientific protocols. The Biotech tropicana, Inc "small tech, big solution” concept aims to establish feasibility of otherwise not attainable technology development oriented research activities, in the resource-poor settings, through cost and complexity reductions, while improving quality.

 

 

Our card board model carries numerous advantages compared to a "real” PCR laboratory. The data in the simulated model are objective, since they are derived from objective scientific data. The model is cheap and simple. In part1, we re-construe a basic PCR facility for application of conventional PCR, using manual sample preparation protocols. Since its inception in the early 1980s the PCR technology has evolved from manual to automation and, from immobile to mobile stations. Different systems developed by different companies introduced automation at each of the four levels of a conventional PCR. [6] Part 1.2 of this work discussed variants of the conventional PCR facility.

 

Modeling in the biological and chemical sciences were previously proven powerful methods by pioneers such as Linus Pauling, James Watson, and Francis Crick. [7]. Here we aim to demonstrate the applicability of the modeling concept in extreme poverty reduction, in the resource-poor settings. The experimental design of the clinical trials of the SMARThivTECHS, comporting a simulation phase, a pre-clinical prototype trial and, a clinical product trial aims to tailor the development of the technologies to the resources and expertise of the resource-poor settings. The simulation phase aims to reduce overall cost of the trial by elucidating potential theory and practice conflict areas, thereby avoiding waste in the "real” clinical trials. This approach is designed in compliance with the United States Millennium Challenge Corporation (US MCC) guidelines, recommending "country ownership” of definition of challenging issues to be addressed in poverty reduction. Resource and expertise levels of the resource-poor settings are by themselves challenges for the resource-poor settings in defining their owned development challenges, for qualification for US MCC assistance. [8;9] Our simulation approach tailored the first step development challenge definition to the resource and expertise of the resource-poor settings, thereby creating ground for qualification for more aid money, by helping the developing world in properly defining their owned development challenges. Here we aim to break the poverty circle at the base, and place the contribution of the developing world in line with the development assistance programs like the US MCC designed in the developed world. The will of the developed world to help the developing world overcome extreme poverty is manifest. However, the bottom line is that it is up to the people of the developing world to come out with ways to overcome extreme poverty, and improve their owned lives. Until the developing world come out with these "new ways”, "more aid money will actually be counterproductive”, and will be used to further empower bad policies, and promote extreme poverty. In these situations, more aid money may actually cause more harm than good to the developing world.

 

 

As part of The SMARThivTECHS tech trials, aiming to develop a PCR protocol tailored to the resource-poor settings, we describe  the basic requirments of a PCR laboratory.

 

 

This publication is part of the Biotech tropicana, Inc  PCR  protocol development project intended for nursing school graduate level readers. We strive  to keep the concept as basic as possible, and the protocols as simple as possible, whithout altering the scientific content. Technically advanced versions are under development at Biotech tropicana, Inc for scientific peer review.   

 

 

 

[1] History and Development of the Polymerase Chain Reaction (PCR), http://www.molecularstation.com/pcr/

 

[2] Theodore E. Mifflin; Setting Up a PCR Laboratory; Department of Pathology, University of Virginia, Charlottesville, Virginia, http://www.biosupplynet.com/pdf/01_PCR_Primer_p.5_14.pdf

 

 [3] University Of Hawai II At Manoa Basic Laboratory Design Guidelines. Available at http://www.hawaii.edu/ehso/industrial/LAB%20DESIGN07.pdf . Accessed December 5, 2010.

 

 [4] Lo et al. 1988. False-positive results and the polymerase chain reaction. Lancet 2: 679.

 

[5] Kwok S. and Higuchi R. 1989. Avoiding false positives with PCR.Nature 339: 237–238.

 

[6] PCR Station. Available at Pcr station, http://www.pcrstation.com/ . Accessed January 7th 2010.

 

[7] Biotech tropicana, Inc. SMARThivTECHS Trials. Available at http://btitechtrials.ucoz.com/publ/smarthivtechs_tech_trial_daily_activities_day_1/1-1-0-2  .Accessed December 10, 2010.

 

[8] David Nummy International Consortium on Government Financial Management. Available at http://www.icgfm.org/downloads/2004miami/nummy.pdf . Accessed Dcember 7, 2009.

 

[9] Available at http://www.kas.de/wf/doc/kas_18129-544-2-30.pdf  . Accessed January 8, 2010.