PCR and qPCR technique

The polymerase chain reaction (PCR, polymerase chain reaction is a technique that was developed in the 1980s by Kary Mullis (Bartlett & Stirling, 2003). It is based on the amplification of specific DNA fragments, allowing millions of copies of these fragments to be obtained from a small sample. PCR is undoubtedly the most important and revolutionary technique in the field of genetics and molecular biology and its impact has been such that Kary Mullis was awarded the Nobel Prize in Chemistry in 1993. Today it is a very common and versatile technique having innumerable applications in genetic analysis in fields as varied as medicine, autopsies, forensic investigations, archaeology or other basic research in molecular biology.

PCR simulates in a vial what happens during cell replication in which an enzyme, DNA polymerase, acts to synthesize two new strands of DNA from another strand that functions as a template. A fundamental step in the experimental design is the choice of primers ( primers The primers are artificially synthesized oligonucleotides (small DNA fragments). Primers are artificially synthesized oligonucleotides (small DNA fragments) with a size of 20-30 base pairs that are chosen during the experimental design to delimit the DNA fragment to be amplified. These primers are characterized by being highly specific, amplifying only the region of interest. Thus, for each DNA fragment to be amplified, a pair of primers is required: one complementary to the 5′ end of one DNA strand and the other complementary to the 3′ end of the other strand ( Figure 1). Además de seleccionar el fragmento a ser amplificado, los iniciadores son el punto de partida a partir del cual la ADN polimerasa iniciará la síntesis de nuevas cadenas.

Before performing the PCR reaction it is necessary to obtain DNA from the samples to be analyzed. DNA can be extracted from any sample containing biological material: blood, saliva, soil, water, etc. This step is critical to obtain samples of sufficient quality for subsequent PCR analysis. DNA extraction from environmental samples is particularly complicated due to the presence of numerous inhibitors of the PCR reaction, such as humic and fulvic acids, as well as other organic substances, metal ions or chemical impurities that precipitate together with the DNA (Desai et al., 2010).

The PCR reaction is performed in equipment called thermal cyclers that subject the samples to different temperatures corresponding to three phases of the reaction: denaturation, alignment and extension (Figure 1).

Thus, the PCR reaction starts with denaturation in which the double helix of the extracted DNA is separated by heating at a high temperature (95 °C), producing single strands that remain as a template for the synthesis of new complementary DNA strands. Once the strands are separated, the temperature is lowered to 40-72 °C, which allows alignment and specific binding of the primers at the corresponding points on the DNA strands. Finally, extension takes place at 68-72 °C where the DNA polymerase enzyme synthesizes the new strands from the primers using the single strands as template. These three steps of the reaction correspond to one cycle and are repeated successively until about 30 cycles are completed. The amplified fragments increase their concentration exponentially as each new copy serves as a mold in the consecutive cycles, thus obtaining millions of copies of the DNA fragment of interest. In case of absence of detection of the amplified product and excluding the hypothesis through a positive control of a reaction failure due to the presence of inhibitors or reagent deficiency, it can be concluded that the sample does not contain the DNA region of interest.

The detection of the presence/absence of DNA fragments is a key tool allowing, for example, to confirm the existence of certain species of microorganisms as pathogens for human health or as the microorganisms responsible for the degradation of a contaminant compound. In this way, it is possible to identify certain dangerous pathogens or to evaluate the effectiveness of a microbiota in bioremediation processes.

Quantitative PCR (qPCR of quantitative PCR or real-time PCR is a technique that combines DNA amplification and detection in a single step by correlating the amplified PCR product in each of the cycles with a fluorescence signal. This technique allows visualization of the reaction in real time and precise quantification of the DNA of interest present in the sample.

The qPCR reaction is performed in a thermal cycler that includes an optical unit capable of detecting fluorescence signals (fluorometer). The fluorometric system consists of an energy source that excites the fluorophores (functional groups of a molecule that make the molecule fluoresce). fluorescent) at a certain excitation wavelength and a detection system that measures the signal emitted at a certain emission wavelength.

Thus, the qPCR reaction follows the same process as a normal PCR reaction with cycles of denaturation, alignment and extension. However, as the DNA fragments are amplified, the fluorophore compound binds and activates, increasing the fluorescence signal emitted.

In the data analysis, the Ct (cycle threshold is the most important parameter in the quantification of DNA by qPCR. The Ct value is the number of cycles required for a significant increase in fluorescence to occur with respect to the baseline signal, i.e. it is when the fluorescence signal passes a certain threshold (Figure 2). Este parámetro es inversamente proporcional a la cantidad inicial del ADN de interés, ya que cuanto mayor sea la cantidad de ADN presente en una muestra, menor será el número de ciclos (valor Ct) que se requiere para alcanzar el umbral. De esta forma, a partir del valor Ct se puede cuantificar la cantidad de ADN.

The analysis of the presence and abundance of DNA by qPCR is a sensitive analysis. It is necessary to have a previous experimental design that optimizes the generation of data and analysis of results, including all the necessary controls to ensure the specificity of the amplification products, as well as to carry out triplicates to certify the reproducibility of the results.

qPCR applied in bioremediation

The remediation of contaminated soils and waters using microorganisms is nowadays widely accepted as a technically, economically and environmentally sustainable strategy. That is why, to date, 50% of the techniques proposed to remediate contaminated sites worldwide belong to the area of bioremediation.

Molecular methods such as PCR and qPCR offer the possibility of knowing the diversity of the microbial population and its different metabolic capacities during biodegradation processes, making it possible to identify the predominant microorganisms actively involved in the bioremediation of various pollutants (Morelli et al. et al., 2015).

The use of the qPCR technique in bioremediation projects provides accurate information for both the design and approach phase of the remediation strategy and the monitoring phase of the contaminated site remediation process.

In this context, qPCR makes it possible to verify the existence and sufficient quantity of specific populations of microorganisms that are responsible for the degradation of the target pollutant, as well as to quantify the genes that produce the enzymes that are key in the metabolic pathway of degradation of the target pollutant. Based on this information, the need to apply a biostimulation or bioaugmentation treatment will be decided in order to successfully develop the remediation process.

Before starting the bioremediation process at full scale, a previous microcosm study is necessary to determine the feasibility and effectiveness of the treatment options proposed under the existing conditions at the site. This study allows to verify if an effective degradation of the contaminant takes place and to rule out possible inhibitions of the microorganisms involved in the process due to the nature and composition of the matrix where the contamination has taken place. The application of the qPCR technique during the microcosm test allows monitoring the evolution of the microbial community and of the enzyme-producing genes involved in the metabolic pathway of the contaminant.

Thus, during the bioremediation process, the populations and genes involved in contaminant degradation can be monitored using this technique. Their evolution will indicate if the treatment is working properly or if there is a limitation in some critical step of the metabolic pathway that, once identified, can be addressed and resolved quickly to maximize the rate of contaminant removal.

The monitoring of specific species during a degradation process is of key importance in those metabolic pathways where the degradation of one of the parent contaminants or intermediates is carried out exclusively by a single species of microorganism. The absence of that species would therefore result in the accumulation of that intermediate and thus block the complete mineralization of the contaminant to harmless products. A clear example of this scenario occurs during the anaerobic biodegradation of chlorinated compounds. This process involves several microbial species that successively transform perchloroethylene (PCE) into trichloroethylene (TCE), TCE into cis-1,2-dichloroethylene (cis-DCE), then into vinyl chloride (VC) and then into vinyl chloride (VC). vinyl chloride) and, finally, in ethylene (Figure 3). El compuesto VC es aún más tóxico que el PCE y TCE, así que evitar la acumulación de este producto intermedio en la ruta de degradación es de vital importancia para no aumentar la toxicidad asociada a la presencia de compuestos clorados en el emplazamiento. Este paso es crítico ya que solo las especies pertenecientes a los géneros Dehalococcoides cuentan con las enzimas necesarias para llevar a cabo la transformación completa de VC en etileno (Lee et al2008, Manchester et al., 2012).

Thus, the qPCR technique represents a fundamental tool in the decontamination of chlorinated compounds as it allows quantifying the copy number of the key species involved in the different steps of the metabolic pathway and monitoring if there is a growth of their population to ensure the complete degradation of PCE to ethylene.

Therefore, the qPCR technique is a tool that provides valuable information about the microbial community and its metabolic capacity. Its application during the design and monitoring of the microbiological treatment allows the detection of deviations that can be quickly corrected, thus contributing to the success of bioremediation.

Bibliography

Bartlett, J., D. Stirling (2003) A Short History of the Polymerase Chain Reaction, Methods in Molecular Biology, 226, pp. 3-6.

Desai, C., H. Pathak, D. Madamwar (2010) Advances in Molecular and “-Omics” Technologies to Gauge Microbial Communities and Bioremediation at Xenobiotic/Anthropogen Contaminated Sites, Bioresource Technology, 101(6), pp. 1558-1569.

Lee, P. K., T. W. Macbeth, K. S. Sorenson, Jr, R. A. Deeb, L. Alvarez-Cohen (2008) Quantifying Genes and Transcripts to Assess the In Situ Physiology of “Dehalococcoides” spp. in a Trichloroethene-Contaminated Groundwater Site, Applied and Environmental Microbiology, 74(9), pp. 2728-2739.

Manchester, M. J., L. A. Hug, M. Zarek, A. Zila, E. A. Edwards (2012) Discovery of a trans-Dichloroethene-Respiring Dehalogenimonas Species in the 1,1,2,2,2-Tetrachloroethane-Dechlorinating WBC-2 Consortium, Applied and Environmental Microbiology, 78(15), pp. 5280-5287.

Morelli. I. S., B. M. Coppotelli, L. Madueño, M. T. Del Panno (2015) Bioremediation in the Post-Genomic Era, Revista Química Viva, 1(14), pp. 26-35.