The polymerase chain reaction (PCR)

2 Literature Review

In this literature review, the polymerase chain reaction (PCR) method is further described and the validity of this technique in forensic science is established. The principles of fluorescence as they are applied to PCR are also discussed in this chapter, together with some discussion of some mechanisms of action of fluorescent dyes and the limitations faced by the researcher in determining the types of dyes used in this study. Finally, the agarose gel electrophoresis procedure used to separate proteins is described and discussed in the context of this study.

The polymerase chain reaction (PCR)

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2.1 The Polymerase Chain Reaction Technique

In general, PCR and the variants to the technique which have been developed over the years follow a similar general process: primers which contain sequences complementary to the target region are used to specifically select the region to be amplified. The DNA polymerase enzyme is used to catalyse the polymerisation of the deoxyribonucleotides into forming a strand of DNA. Thermal cycling is used throughout the process in which the reaction is repeatedly heated and then cooled, where the heating cycles allow DNA melting to occur which breaks up the DNA strand into deoxyribonucleotides while the cooling cycles allow for enzymatic replication of the DNA and the subsequent re-polymerisation of the deoxyribonucleotides into DNA strands. This process allows for an exponential multiplication of the DNA section to be tested and generates a large number of copies of the DNA sequence which subsequently allows for many tests to be performed on the DNA strand even from a limited amount in the original sample.
PCR has wide applications in forensic science. In practical situations, PCR has been applied to identifying war victims from mass graves (Primorac et al, 1996), determining the species of different species from various samples (Murray, McClymont and Strobeck, 1995), or determining the specific breed of dog from a forensic sample – all dogs are the same species although generations of selective breeding has created a single species which can display radically different phenotypes (Halverson and Basten, 2005). While restriction fragment length polymorphism (RFLP) techniques had been used in the past, PCR has the advantage of being fast, highly discriminative and can also be used to process multiple samples at once (Luftig and Richey, 2001). For example, the amplification of variable number of tandem repeats (VNTR) is a widely used technique described in Kasai, Nakamura and White (1990) that allows for the amplification from a very small quantity of genomic DNA obtained from forensic samples and which can be combined with DNA profiling techniques to identify a particular individual. The PCR technique is sufficiently adaptable and powerful that it can be used for DNA samples that have been chemically contaminated (Wilson et al, 1995) and even adapted for samples of blood which may contain a contaminant that inhibits the PCR process (Akane et al, 1994). However, it should be considered that there may in fact be many inhibitors that prevent the success of the PCR and many other factors that should be considered when performing a PCR.
While the commercially available starting materials and reagents may be pure and of high quality, PCR can still fail and there are two critical components that require discretion from the researcher. Firstly, the nucleic acid template needs to be of sufficient quality and should have no DNA polymerase inhibitors although the PCR technique can be quite tolerant of impurities compared to other molecular biology techniques. Secondly, the selection of the oligonucleotide primers is critical. This is not a trivial problem particularly when more complex techniques such as multiplex or nested PCR are used (Dieffenbach, Lowe and Dveksler, 1993). Nevertheless, the use of computer algorithms allows primer selection to be simplified and can include commercial software (for example, Invitrogen by Life Technologies (2010)) or free software (National Institutes of Health, 2011).
Having established the importance of ensuring that the oligonucleotide primers chosen are suitable for analysis, this review now turns to the literature that establishes that the choice of the polymerase enzyme also has an effect on the efficiency of the PCR. In the PCR technique, the DNA polymerase most frequently used is known as the Taq polymerase. The Taq polymerase is used specifically because a DNA polymerase which is able to withstand the high temperatures encountered in the heating cycles of the PCR technique which allow DNA melting but which ordinarily cause protein denaturation. The selection of the Taq polymerase which is obtained from a thermophilic bacteria known as Thermus aquaticus, is described in Saiki et al (1988). However, the Taq polymerase enzyme lacks 3′ to 5′ exonuclease proofreading activity which contributes to an error rate of approximately 1 in 10,000 nucleotides (Tindall and Kunkel, 1988). DNA polymerases which exhibit the 3′ to 5′ exonuclease proofreading activity exist; for example, the Pfu polymerase possesses that ability which results in an error rate of approximately 1 in 1.3 million nucleotides, and which also exhibits thermostability, but which comes at the cost of being slower than the Taq polymerase (Cline, Braman and Hogrefe, 1996). The choice of DNA polymerase to be used in a specific experiment therefore becomes a complex trade off between the accuracy of replication, the speed of the enzyme used and cost.
Therefore, when considering the sensitivity of the PCR reaction, the issue of optimization of the PCR procedure becomes a critical one. The trade offs involved in selecting the DNA polymerase to be used in the experiment have already been discussed but several other factors are critical to the success of an experiment involving PCR. For one, because of the high sensitivity of the PCR procedure, it is particularly susceptible to contamination. While this is not a concern in many research settings, if the PCR is designed to be very sensitive or if the presence or absence of amplification of a target sequence will be used in diagnosis, contamination needs to be eliminated for the results to have any significance. The most troublesome source of contamination is known as “carryover” contamination where DNA generated by previous PCR amplification of the target sequence contaminates the sample. Therefore, controls are essential to detect carryover contamination and the procedures to prevent this, and other, types of contamination are discussed in detail in Hartley and Rashtchian (1993) and Scherczinger et al (1999).
The Taq polymerase is a magnesium-dependent enzyme and the optimization of Mg2+ is critical in a PCR procedure that involves the use of this enzyme (Markoulatos, Siafakas and Moncany, 2002). The specific concentration of Mg2+ is dependent on the concentration of dNTP, the specific template DNA and the composition of the sample buffer. On the one hand, excessive Mg2+ stabilizes the DNA double strand and inhibits the complete denaturation of DNA which reduces the yield and can also stabilize spurious annealing of the primer to incorrect template sites which decreases specificity. On the other hand, inadequate Mg2+ will lead to a decrease in the amount of product. Since the appropriate concentration of Mg2+ and dNTP cannot easily be determined before the experiment has begun, the recommendations are that varying concentrations of both Mg2+ and dNTP be tested with the specific reagents for use in the experiment so that the optimal concentration can be determined (Henegariu, Heerema and Dlouhy, 1997).

2.2 Fluorescence and its role in quantitative PCR

Although standard PCR techniques are effective in amplifying the amount of DNA that has been recovered from a sample of DNA, a variant on the technique to actually quantify the DNA that has been generated is known as quantitative PCR. While the specific techniques may differ slightly, all quantitative PCR amplifications are performed in the presence of a DNA-binding fluorescent dye with the implicit assumption that the fluorescence detected is directly correlated to the amount of double stranded DNA present in the amplification reaction (Higuchi et al, 1992). Through the application of specific fluorescent dyes that bind to specific products of the reaction, continuous monitoring of DNA amplification throughout the PCR reaction can be achieved (Wittwer et al, 1997). One of the first applications of fluorescence can be found in Smith et al (1986) in which a method was developed where differently coloured fluorescent dyes would bind specifically to those reactions that are specific for the bases C, G, A, and T, and the separated fluorescent bands of DNA were detected and analysed. Significantly, the use of fluorescent dyes allows the monitoring of PCR reactions in real time and can be very sensitive, specific and reproducible although the experiment must be designed carefully. The process generally requires the comparison of a biological sample against a standard curve of known initial concentration when absolute quantification is required, or comparing the expression of a specific gene to an internal standard when relative expression is required instead (Bustin, 2000).
One of the fluorescence based techniques commonly used is the colour complementation assay described in Chehab and Kan (1989). In the colour complementation assay, two or more DNA segments are amplified with fluorescent oligonucleotide primers such that a colour, or combination of colours, can be measured and then used to deduce the quantity of each DNA segment present in the sample. Several commercial examples of these fluorophores can be found in the Applied Biosystems fluorescent NHS ester dyes FAM and ROX which conjugate to specific primers, respectively, to give green and red PCR products (Carozzi et al, 1991).
A description of the chemistry involved in fluorescence based quantitative real time PCR procedure is provided by Grove (1999). A probe is first designed to anneal to the target sequence between the forward and reverse primers. At the 5′ end, the probe is labeled with a reporter fluorochrome (6-FAM described in Grove (1999)), and a quencher fluorochrome (TAMRA) is added to any T position or the 3′ end. As long as the fluorochromes are on the probe, the quencher molecule stops all fluorescence by the reporter. However, since Taq polymerase extends the primer, the nuclease activity of Taq polymerase degrades the probe which releases the reporter fluorochromes into the solution and therefore the amount of fluorescence which can be detected is due solely to the fluorochromes released in this process and this is subsequently proportional to the amount of product generated per cycle. As each cycle of amplification proceeds, new rounds of hybridization occur and additional fluorochromes are released from the probes which result in higher fluorescence and correspond to an increase in the target DNA molecules (Marras, 2006).
The selection of the appropriate fluorescent dye and associated quencher is therefore important in generating viable results in the quantitative real time PCR technique. For example, ethidium bromide has been used as a fluorescent dye to detect the presence of double stranded DNA but can only produce semi-quantitative results in PCR at best. This is because ethidium bromide staining saturates at low levels of DNA and is therefore not sufficiently sensitive for accurate results (Martin, 2008).
In this study, a dye set based on the blue dye FAM is used. In the process of sequencing with dye primers, four different types of dyes are normally used in a single set and the dyes are selected so that they fluoresce at different wave lengths of light – blue, green, yellow or red. The dye sets are normally chosen so that the chemical properties of the dyes complement one another: for example, each dye in a set consisting of 5-FAM (blue), JOE (green), TAMRA (yellow) and ROX (red) dyes will attach to a different 3’ terminal dideoxynucleotide. By examining the wavelengths at which the sample fluoresces when excited by light, the colour of the fluorescence can be analysed to determine the final base in the dideoxynucleotide: whether A, G, C, or T.
The detection of fluorescence is dependent on the type of instruments used. In most modern instruments, fluorescence detection is done through a charge-coupled device (CCD) which is able to detect fainter and more transient light from sources than the human eye or even photographic plates are able. Nevertheless, in most instruments, the light emitted is dependent on the type of fluorescent die used and the wavelength of light emitted when the sample is excited through the use of a laser. In the case of the Applied Biosystems ABI PRISM 310 Genetic Analyzer, which is the instrument available to the researcher in this study, an argon ion laser is used and results in maximum fluorescence emission and excitation wavelengths when various fluorescent dyes are used (Applied Biosystems, 2000). This also introduces the limitation of the types of fluorescent dyes that can be used in PCR experiments since only a number of dyes are supported by the instrument used. However, the different emission and excitation wavelengths of each of the dyes can be used in a multiplexing perspective to detect a broad range of results; for example, the wavelengths of light emitted when various dyes which emit at different wavelengths can be used to deduce the quantity of the required DNA fragment available in the sample.
Fluorescence has been used as a method for DNA analysis in previous work. Chen et al (1997) reported the use of a fluorescence-based assay method, the template-directed dye-terminator incorporation (TDI) assay, to detect mutations in various human genes. Chen et al (1997) began with a sample of total human DNA, used PCR to amplify the required DNA fragments and performed the primer extension reaction to detect those DNA fragments that had mutated. The experiment found that, by using two dyes – ROX and TAMRA – it was possible to detect mutations to a high degree of accuracy by detecting the changes in fluorescence intensity.
Baele et al (2001) in conducting tests of reproducibility of a fluorescence-based assay method, attempted to distinguish between different strains of Streptococcus bacteria in three different labs. The technique examined was known as tRNA intergenic length polymorphism analysis (tDNA-PCR) and involved the amplification of DNA primers to which fluorescent markers had been attached. The results of the experiments showed that the methodology was highly specific and could easily identify the species of bacteria, while the results were found to be identical across the three different laboratories and therefore highly reproducible.

2.3 Fluorescence Technique

Fluorescence techniques involve various aspects of the concept. For instance, Kasten (1993) describes fluorescence process as one of the concepts of the techniques. In this process fluorescence is described as a three-stage process occurring in certain molecules, which are generally polyaromatic hydrocarbons or heterocycles. The stages include excitation, excited-state lifetime, and fluorescence emission. In all the three stages, there are specific characteristics of properties that identify the same in a bid to differentiating the specific stages (Kasten, 1993). For instance, whereas stage three is characterized by emission of photon energy, stage two is described by the conformational changes whilst being a subject of numerous interactions with the possible molecular environments. On the other hand, there is supply of photon energy from an external environment in order to excite the process in the first stage. These stages and their characteristics are very important in defining the process of fluorescence thus developing appropriate software capillarity.
A capillary electrophoresis fluorescence detector usually occur on-capillary through different aspects such as deuterium, xenon arc lamps, and tungsten. Capillary electrophoresis has been identified to benefitting significantly from various devices especially the micro-fabrication of the same (Valeur, 2002). Such devices are known to have performed efficient separations in a routine, fast, and highly effective way. Other than helping in miniaturization, micro-fabrication techniques or methods have been identified to be helpful in producing various complex devices that have high degree of functionality. In addition, these devices have been able to demonstrate the potential within complex DNA microprocessors (Kasten, 1993). Capillary electrophoresis micro-devices have been identified as fabricated through bonding and glass substrate technology. In any case, such devices have also been identified as being able to utilize off-chip photomultipliers especially for various detections by fluorescence (Albani, 2007). It is also possible that through on-line detection techniques and methods, it is possible for capillary electrophoresis to make portable DNA diagnostic instruments to be practical unlike in other many cases when such diagnosis have not been practical in the past.
Researchers have also identified the fact that capillary electrophoresis in connection with laser-induced fluorescence (LIF) provide utmost needed speed, sensitivity, as well as resolving power. In addition, such combinations have been capable of giving analyses of a wider number of solutes. Single molecule detection as well as DNA sequencing coupled with analysis of polymerase chain reaction (PCR) products are examples that accrue from capillary electrophoresis. According to Albani (2007), other examples resulting from combination of capillary electrophoresis and laser-induced fluorescence (LIF) are single cell analysis as well as analyses of proteins and small solutes. The past years have been influential in enhancing capillary array electrophoresis (CAE) especially in sequencing human genomes and drug screening (Kasten, 1993). Therefore, methods based on CE and LIF have the likelihood to be suitable especially in sequencing more human genomes meant in making comparison as well as diagnosis and testing of genetics (Kobayashi, Ogawa, Alford, Choyke, and Urano, 2010). Proteomics that represent major pressure for purposes of development even higher thoughput as well as robust analytical technologies have been associated with combination of the capillary electrophoresis (CE) and the laser-induced fluorescence (LIF).
Chemicals are either mono-intercalating or bis-intercalating dyes. With respect to intercalating dyes, chemicals are weakly fluorescence thus having the potential of strong fluorescent upon forming complex-grounds. Such complex grounds employ CE and LIF due to the low back-ground. Analysis of DNA provides a demonstration of intercalating dyes within the background electrolyte also known as polymer solution. From the complexity of this electrolyte, it is possible to obtain double-stranded DNA within the process of analysis. A minor division of intercalating dyes is the mono-intercalating dyes as well as the bis-intercalating dyes (Kricka and Fortina, 2009). These forms of dyes are essential in understanding the concept of DNA. Analysis of DNA is very essential in locating the different dyes that are present within the fluorescence techniques. Since various techniques are used within the fluorescence process, there is a possibility of coming up with different forms and minor forms of dyes (Kricka and Fortina, 2009). Such forms and minor forms of dyes are only identifiable through the fluorescence techniques available within the contemporary and classical world. Applications of fluorescence are very vital in ensuring that DNA testing and diagnostics are enhanced.

2.3.1 Exciting of Fluorescence in Capillary Electrophoresis

Exciting fluorescence involves supply of photon energy from an external environment in order to excite the process in the first stage. In capillary electrophoresis (CE), exciting fluorescence requires selection of an efficient and capable wavelength especially during the grafting of monochromator (Valeur, 2002). Grafting monochromator is a technique applicable in selecting the proper and efficient wavelength, which is very essential in providing appropriate photon for exciting stage. Photon provided from the exciting wavelengths arising from proper selection after applications of the grafting monochromator is essential in giving out the necessary energy for the same process. Excitation process within fluorescence in the first stage is very essential in enhancing the process of obtaining proper photon, which becomes very vital in other functions. Once excitation stage is fulfilled, it becomes easier for the capillary electrophoresis (CE) and other essential processes to take place (Albani, 2007). This enables the fulfilment of various other objectives of capillary electrophoresis.

2.3.2 Emission

Emission is the last stage of fluorescence. This stage is usually characterized by emission of photon energy. In most cases, the emitted photon energy arises from the selected wavelength through the process of grafting monochromator (Joo, Balci, Ishitsuka, Buranachai, and Ha, 2008). It is important to note that emission of photon energy especially within the capillary electrophoresis occurs after attaining the objectives of the same. Photon energy emitted in this stage has different wavelengths, which are determined and selected solely by the grafting monochromator (Kasten, 1993). Differences in photon wavelength is also determined by the function of the energy especially in regards to attaining objectives of capillary electrophoresis (CE) that revolves around testing of the DNA (Albani, 2007). This is very influential in making sure that there is enough energy to carry out all the processes involved within the fluorescence technique especially within the capillary electrophoresis (CE). Therefore, there is evidence that emission stage within the technique of fluorescence is very likely when there is enough grafting monochromator, which provides adequate energy in terms of photon for doing the same functions.

Bibliography

Albani, J.R., 2007, Principles and Applications of Fluorescence Spectroscopy, Wiley-Blackwell, New York
Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. and Ha, T., 2008, “Advances in single-molecule fluorescence methods for molecular biology,” Annu Rev Biochem, Vol. 77:51–76.
Kasten, F.H., 1993, “Introduction to Fluorescent Probes: Properties, History and Applications” in Fluorescent and Luminescent Probes for Biological Activity, Academic Press, W.T. Mason, Ed.
Kobayashi, H., Ogawa, M., Alford, R., Choyke, P.L. and Urano, Y., 2010, “New strategies for fluorescent probe design in medical diagnostic imaging,” Chem Rev, Vol. 110:2620–2640.
Kricka, L.J. and Fortina, P., 2009, “Analytical ancestry: “Firsts” in fluorescent labeling of nucleosides, nucleotides, and nucleic acids,” Clin Chem, Vol. 55:670–683.
Valeur, B., 2002, Molecular Fluorescence: Principles and Applications, John Wiley and Sons, New York.

By admin in Essays on October 30, 2015