*Corresponding author:Alicia García Falgueras, University Researcher, Netherlands Institute for Neuroscience, Spain
How to cite this article:Alicia García F. Polymerase Chain Reaction (PCR) and How Temperature Modifies Nucleotide Pairings. Curr Trends Biomedical Eng & Biosci. 2021; 20(1): 556027. DOI:10.19080/CTBEB.2021.20.556027
Polymerase Chain Reaction (PCR) is a molecular technique more suitable for accurate diagnoses because of its specificity in targeting and identifying specific pathogens. PCR technique is reliable to detect the genetic presence of the virus in the host since the very beginning of the infection, no matter how the immune system of the person might react. Moreover, the samples for the test contains the DNA information during quite a long time since the extraction. Programmable thermal operation cycler for controlling heating and cooling is the procedure to denaturation into two single strands, annealing and extension reactions of the DNA. The PCR is certainly having a clinical approach, because do not emphasized on how the immune system of the patient might reacts to the infection, only on whether the specific virus is present or not for the accurate diagnoses and subsequently proceed with an appropriated medical treatment to restore health.
To identify genotypes characteristics for basic and ethical clinical research the PCR has been of extended use [1-3] and remains by far the most widely used. Differences in genotypes of certain population with a) a mutation can be detected with PCR  or b) to described and define a specific population depending on their hetero or homozygosis for certain genes, the PCR procedure is applied [2,3]. Genome of most of species have been publicly disclosed and genome sequencing can be used by researchers to design primers for PCR for their experiments to probe fragments bases pair (bp) as simple primer sequences (5´- 3´), length polymorphisms (SSPLs, microsatellites), etc. [2,3]. PCR primers are between 18 and 30 nucleotides in length and should preferably include a guanine + cytosine pair (GC). The Thermus acuaticus (Taq DNA polymerase, originally isolated by Chien et al., 1976 ), Pyroccocus furiosus (Pfu DNA polymerase), Thermus thermophilus (Tth polymerase, for RNA templates) and Thermococcus litoralis (VentTM DNA polymerase) are the most commonly enzymes, thermophilic microorganisms, used to copy the DNA: they are high heat resistant (95 oC) and able to synthesize nucleotides which are the four basic bases-molecules essential for life (A, T, C, G) and are amplified each successive cycle doubles the amount of DNA product . Since its original description in 1985 by Kary Mullis , the PCR technique has evolved into a multitude
of varied methodologies. Because of the natural components of the PCR, this technique has shown a surprising robustness: 1) the impressive ability of almost all oligodeoxynucleotides to bind tightly and discriminating to their specific complementary nucleic acid sequences, 2) the fact each component is necessary and sufficient for the reaction, acting each one in the same probability in a cascade and 3) the evidence that the mechanism is related to the branching structure of its robust propagation and amplification .
The abundant and exponential amplification of a very small amount of template DNA are the constant theoretical and technical aspects of the PCR, to analyze followed by specific enzymatic restriction digestions (i. e. Sau96I; HhaI) according to the nucleotide sequence which needs to be localized. Whole samples of blood are lysis buffered to open the cells, filtered, rinsed, washed several times, till DNA elution to be amplified is isolated via centrifuged by spinning the different solutions through the deposit assembly. This sedimentation according to their molecular weight is the reason why they are easily distinguished on the three dimensional matrix of agarose gel electrophoresis. Due to their different biomolecular sizes they are vertically possible to see and distinguish under UV light [3,7,8]. Programmable thermal operation cycler for controlling heating and cooling is the procedure to denaturation to two single strands, annealing and extension reactions of the DNA (Figure 1). The following protocol conditions is commonly used: 95 oC, 50 seg., 57 oC 50 seg., 72 oC,
50 seg., (from 25-40 cycles, total 2 hours) with a final extension at
72 oC for 10 min. [3,7], but temperatures can be slightly modified
for optimizing assay conditions, as well as reagent conditions and
incubation times. The annealing temperatures varies from 57 oC
to 64 oC . This is how sensitivity to increases and decreases of
temperature is such a delicate issue for the efficiency and efficacy
of this technique. Sensitivity of PCR technique for coronavirus
diagnosis in contexts of a high prevalence is 86 %, having a
specificity of 96 % .
PCR as a molecular technique is more suitable for accurate
diagnoses because of its specificity in targeting and identifying
specific pathogens . Testing nucleotic acids amplification
(NAAT) is the primary method of COVID-19 diagnoses and it
has been recommended by the WHO for the better patients tests
. Samples are not only whole blood as described, but also
respiratory samples (nasal and throat swabs), reliable 8 days after
symptom onset. SARS-CoV-2 virus has four structural proteins
which can be identify with optimized PCR and RT-PCR protocols.
The coronaviruses have a genome size of about 30 kilobases
(kb) . Several kits have been approved for detecting SARSCoV-
2 however, the reverse transcriptase PCR (RT-PCT) and the
Real-Time Reverse transcriptase PCR (rRT-PCR) with respiratory
samples are the most commonly method used for diagnoses to
detect the virus genetically (Figure 2). Due to the rapid spread
of coronavirus diseases a reliable detectable method is needed
for an accurate diagnose . The sequenced RNA of the virus
which belongs to specific synthesis of proteins, is transcript
into complementary DNA (cDNA) strands and subsequently the
selected fragments of cDNA is amplified . After increasing
the number of DNA molecules, detection and visualization of
nucleotides combinations are usually done by methods like
agarose gel electrophoresis .
PCR technique is reliable to detect the genetic presence of
the virus in the host since the very beginning of the infection and
first symptoms appear, no matter how the immune system of the
person might react. Moreover, the samples for the test contains
the virus DNA information during quite a long time since the
extraction (8 days at least). This technique is delicate and requires
specific protocols and temperatures to success in the location but
errors are minimum due to the exponential amplification of the
DNA sequences. The PCR is certainly having an ethical clinical
approach diagnosis in contexts of a high prevalence of infections.
That is due because PCR does not emphasized on how the
immune system of the patient might reacts to the virus, but only
on whether the specific virus is genetically present or not for the
accurate diagnoses. Subsequently after virus detection genetically
it is possible to proceed with an appropriated medical treatment
to restore health on affected patients.