Polymerase chain reaction (PRC), is a technique used in the replication of part of a DNA strand in vitro (outside an organism). The PCR process is dependent on the availability of a DNA primer specific to the region that should be amplified and Taq polymerase activity (Hern à ndez-crespo et al., 2016). The basic steps of PCR are denaturation, annealing, and extension. The denaturation step is undertaken at a temperature of 96 °C (Huggett, Cowen, & Foy, 2015). During the denaturation step, the double helix DNA is separated into two single strands. The annealing binds the primer to its complementary DNA template at a temperature of about 55 °C . The extension stage relies on Taq polymerase to join and extend the amplicons forming a new DNA strand. The components of a PCR mix include a DNA template, DNA polymerase, primers, four types of deoxyribonucleotide triphosphates, and a reaction buffer. The DNA template is the specified DNA sequence which one wants to amplify. DNA polymerase is vital in initiating and sustaining the replication of the target DNA sequence. Primers are essential in the PCR mix since they amplify the target DNA sequence in the genome (Tran, Pham, & Trinh, 2015). The four deoxyribonucleotide phosphates are important because they are the precursors for the new DNA strand. Finally, a reaction buffer is introduced to stabilize the pH.
The volumes of the components of the PCR mix are 40 μL of 10x PCR reaction buffer (which includes 25 mM MgCl2), 8μL of 10μ M dNTP, 32 μL of Sterile H2O, and 6ml of MgCl2 (Lorenzo, 2012). The choice of volumes of the PCR c omponents is base on optimization of the replication of the target DNA sequence by presenting conducive conditions. Also, 16s rRNA is used in PCR because the sequence is highly conserved among various species of archaea and bacteria (Lu et al., 2015). The 16s rRNA gene is amplified to increase the concentration of contaminating bacterial DNA to permit a better examination of the species. Before conducting PCR, normal DNA is diluted to negate the effect of inhibitors. Kit DNA is not diluted since it acts as the control.
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Preparing a master mix is vital in preventing pipetting errors. Principally, pipetting error can hinder the reproducibility of the DNA template. Principally, Taq DNA polymerase is a PCR component purified from Thermus aquaticus . DNA polymerases are used in a PCR to polymerize nucleotides forming a DNA duplex. The polymerization occurs in a 5’ to 3’ direction. DNA polymerase also has exonuclease activity. Unlike conventional Taq DNA polymerases, Vinantis Taq DNA polymerase is ultra-pure and can promote amplification of up to 20kb. Furthermore, the 10X ViBuffer S promotes amplification of 5 amplicons and above. The Vinantis Taq DNA polymerase is also suitable for TA cloning since it generates amplicons with 3’ dA overhangs. Ultimately, Vinantis Taq DNA polymerase is more effective in PCR compared to a normal Taq DNA polymerase.
Amplification of a DNA strand using PCR can only be accomplished through the activity of the reverse and forward primer. In this regard, a forward primer that marks the beginning of a gene and a backward primer that will begin the DNA sequence that is complementary to the template are vital in the production of amplicons (Frank et al., 2008). In the case of this laboratory experiment, the reverse and forwards primers are the 492r Reverse primer and 27f Forward primer respectively (Fredriksson, Hermansson, & Wil é n, 2013). Moreover, a negative control in PCR is not expected to yield amplicons, while a positive control is expected to produce viable amplicons. The purpose of the negative and positive controls is to test the efficiency of the master mix, primer annealing temperature, Magnesium chloride amounts, and the extension time. According to Salamon (2017), keeping PCR tubes on ice until when the sample DNA is added prevents denaturation of the enzyme. Hence, guaranteeing optimal enzyme activity. The DNA sample is introduced last after all optimal conditions have been established to promote a higher amplicon yield.
References
Frank, J. A., Reich, C. I., Sharma, S., Weisbaum, J. S., Wilson, B. A., & Olsen, G. J. (2008). Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and environmental microbiology , 74 (8), 2461-70.
Fredriksson, N. J., Hermansson, M., & Wil é n, B. M. (2013). The choice of PCR primers has great impact on assessments of bacterial community diversity and dynamics in a wastewater treatment plant. PloS one , 8 (10), e76431. doi:10.1371/journal.pone.0076431.
Hern àndez-crespo, P., Beroiz, B., Casta ñera, P., Ortego, F., Salillas, M. J. C., VEGA, M. L., & V á zquez, C. A. (2016). U.S. Patent Application No. 14/905,211 .
Huggett, J. F., Cowen, S., & Foy, C. A. (2015). Considerations for digital PCR as an accurate molecular diagnostic tool. Clinical chemistry , 61 (1), 79-88.
Lorenz, T. C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. Journal of visualized experiments: JoVE , (63).
Lu, V. T. R., Tharayanil, A., Anderson, J., Tsiatis, A., Bailey, H., Bonham, M., ... & Shak, S. (2015). P032 Central lab HER2 testing by RT-PCR, IHC and FISH in locally HER2-Neg, ER+ IBC with in situ carcinoma.
Salamon, D. (2017). Analysis of Viral Epigenotypes Using Bisulfite Sequencing: A Detailed Protocol for the Crucial Bisulfite Modification and PCR Amplification Steps. In Epstein Barr Virus (pp. 207-213). Humana Press, New York, NY.
Tran, H. T., Pham, Y., & Trinh, P. L. (2015). AB141. Multiplex PCR-based procedure establishment for simultaneous detection of two mutations occurring most frequently in FMS-like tyrosine kinase-3. Annals of translational medicine , 3 (Suppl 2).
