PCR Experiment: DNA Amplification and Analysis

Abstract

PCR is a powerful amplification technique that can be used to generate a large amount of sample from a small amount of starting material. Students were instructed to carry out various experiments that are involved in the restriction, amplification, and visualization of DNA/RNA. The aim of the experiment was to familiarize the students with these various procedures, and results indicated a high level of understanding and practical capability of many of the students. Potential improvements of the experiment are possible due to time constraints preventing perfect results as it would be of utmost importance for future scientists to be aware of such an important developing technology.

Introduction

In the 1980s, a method for in-vitro amplification of specific DNA/RNA sequences using PCR has revolutionized the methods used in molecular biology. PCR has various uses in biology and allows for early diagnosis in many areas of medicine. PCR can easily produce more than a million copies of a specific DNA or RNA sequence by utilizing a simple three-step method (^{Johnson, 1991}). The first step involves the denaturation of DNA strands, the second step anneals primers to the denatured DNA strands. Thirdly, the primers are involved in an extension reaction catalyzed by a thermostable DNA polymerase, and the cycle is then repeated. The polymerase chain reaction has great advantages over other technologies in the same field as it allows direct detection of microbial DNA with high accuracy of known positive pathological specimens (^{Johnson, 1991}). However, it can also detect gene-harboring strains regardless of gene expression, meaning a positive result may indicate a false-positive as it does not indicate an active or expressed gene but rather the presence of it (^{Garibyan and Avashia, 2013}). It can also be a very costly method to run efficiently. The aims of this study were to develop the knowledge and skills of the students involved so that they would know how the polymerase chain reaction is performed and how it is made possible, as well as being able to understand how to utilize all the equipment involved in PCR, including being able to understand the DNA purification process, the gel electrophoresis process, and how to read and understand the final results.

Methods

In order to maintain the best time-frame and be able to complete all the tests within the 3 hours, the tasks were completed in this order: Gel Electrophoresis > Restriction Digestion > PCR > DNA Purification.

PCR

The first step for the Polymerase Chain Reaction is to add the following reagents to a centrifuge suitable tube:

• DNA template: 7.5 ul
• Primer Forward (10 uM, Tm 62 °C): 2 ul
• Primer Reverse (10 uM, Tm 61 °C): 2 ul
• dNTPs (10 uM each): 2 ul
• Buffer (10X): 5 ul
• Taq Polymerase: 2 ul
• Water to fill the tube to 50 ul

Then mix well using a pipette and centrifuge briefly at high speed to mix well. Place the tube into a PCR thermal cycler, and then record the best Annealing Temperature.

DNA Purification

Add 150 ul of Buffer PB to 30 ul of PCR sample and then mix well, place a QIAquick spin column in a 2 ml collection tube. Then apply the PCR-Buffer PB sample to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and then discard flow-through. Place the QIAquick column back into the same collection tube and then wash with 750 ul Buffer PE to the QIAquick column and centrifuge for 1 min at 13,000 rpm. Discard flow-through and place QIAquick back in the same collection tube. Centrifuge for an additional 1 min at 13,000 rpm to remove all residual ethanol from Buffer. Place the QIAquick column in a clean 1.5ml Eppendorf tube. Then add 50 μl of Buffer EB to the center of the QIAquick membrane. Stand the tube at room temperature for 1 min. Centrifuge the tube for 1 min at 13,000 rpm.

Restriction Digestion

Add 5 ul of Plasmid pBluescript template, 2 ul HindIII enzyme, 13 ul of water. Mix well with a pipette and centrifuge quickly at high speed. Place the tube in a 37°C heat block for 30 minutes.

Agarose Gel Electrophoresis

Demonstrators prepared the gel tank and gel tray for the sake of time, as well as loaded the gel tray into a gel tank containing 1X TAE.

Add 3 ul of 6x loading dye, and then aliquot the whole sample into the well. Load the molecular weight marker, and then run the gel for 30 minutes at 160V.

Results

The electrophoresis had varying results from the different wells, but in general, the bands were visible, and comparisons were able to be made. It can also be seen that the setup from other steps such as the restriction digestion were completed correctly as they separated properly on the electrophoresis results.

Agarose gel (1%) electrophoresis results, in well 1 is the molecular weight marker, and in well 9 is our personal results. The Uncut vector is located in well 2. It’s visible that the enzyme restriction was successful as the samples are showing different bands.

Discussion

According to our results, we are able to conclude that the we were able to restrict the DNA in the sample wells somewhat into a predicted result, with the number of bands aligning with our predicted results. The general aim of the experiment was to allow participating students to gain a higher knowledge of all the processes involved in a successful PCR, and how specific regions of DNA/RNA are acquired and how they can be visualized using gel electrophoresis. It seems as though students were able to complete all steps of the practical without many issues which indicates a high success rate, and also that the results acquired for the Optimal Annealing Temperature and the DNA purification steps were successful as seen in Figure 1 as the bands have become quite visible.

As seen in Figure 1, the results indicated that students were able to carry out this experiment with a high rate of success; however, there were multiple errors within the results that displayed either student mistakes or a rush of procedure due to time constraints. As one of the disadvantages of PCR is that it can amplify DNA/RNA contaminants, it allows for very little margin for error, as there will be visible mistakes when viewing the gel electrophoresis results. For example, in well 5 of figure 1, there seems to be a contaminant in their sample and it has been visualized on the gel. It is also visible on the gel that not all samples were restricted properly, and this is most likely due to the time restraints, as the samples most likely needed more time for the restriction enzymes to thoroughly split all of the DNA into the desired amount of sections of basepairs. It would be optimal for the study to be re-conducted with more time for the samples to settle in the future so that there can be more reliable results when visualized, as this would be of high importance in a laboratory environment handling precious samples.

There is a great future for PCR, the combining of various assays to create a higher understanding of various gene combinations (^{Botes et al., 2011}). For example, a study was conducted in order to connect distinct species from the microbial community to specific metabolic processes, which was made possible by combining stable isotope probing with qPCR (^{Postollec et al., 2011}). There have also been academic groups that are attempting to improve PCR diagnostics. For example, an alternative temperature cycling method is being developed by Luke Lee of the University of California, in which LEDs heat a 120nm gold film in contact with a DNA solution in a microfluidic well (^{Son et al., 2015}). This new cycling method could potentially allow for heating from 55°C to 95°C, 30 times in 5 minutes. This is of particular significance because if PCR could be used to identify certain diseases in human beings within just a couple of hours that could mean the difference between life and death, as it would allow for the correct and specific treatment to be acted on incredibly quickly, allowing for better chances of survival (^{Menon et al., 1999}).

References

1. Botes, M., de Kwaadsteniet, M., and Cloete, T. (2012). Application of quantitative PCR for the detection of microorganisms in water. Analytical and Bioanalytical Chemistry, 405(1), pp.91-108.
2. Garibyan, L., and Avashia, N. (2013). Polymerase Chain Reaction. Journal of Investigative Dermatology, 133(3), pp.1-4.
3. Johnson, M. (1991). The Polymerase Chain Reaction: An Overview and Development of Diagnostic PCR Protocols at the LCDC. Canadian Journal of Infectious Diseases, 2(2), pp.89-91.
4. Menon, P., Kapila, K., and Ohri, V. (1999). Polymerase chain reaction and advances in infectious disease diagnosis. Medical Journal Armed Forces India, 55(3), pp.229-231.
5. Postollec, F., Falentin, H., Pavan, S., Combrisson, J., and Sohier, D. (2011). Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food Microbiology, 28(5), pp.848-861.
6. Son, J., Cho, B., Hong, S., Lee, S., Hoxha, O., Haack, A., and Lee, L. (2015). Ultrafast photonic PCR. Light: Science & Applications, 4(7), pp.e280-e280.