Molecular Biology And Polymerase Chain Reaction (PCR) Course

Becoming a professional in this field requires students to understand how DNA is organized into genes and the function and regulation of genes in the context of a genome. Students are expected to understand similarities in gene sequence and function between different organisms, and to contrast this with differences in DNA sequence (genetic variability) between individuals in a population. Understanding theoretical concepts in molecular biology will be strengthened by practical classes that provide students with experience in a molecular biology laboratory and the skills to analyse and report on experiments. In this way, this unit aims to introduce fundamental concepts and methods in molecular biology that can be used by students to develop an understanding of the applications of molecular biology to their own field of interest.

After successful completion of this Unit, students will be able to:

1 Recall common terms and definitions of molecular biology

2 Explain how genomes are organised and how this relates to the control of gene expression.

3 Describe mechanisms of transcriptional and post-transcriptional gene regulation

4 Compare and contrast common approaches to analyse DNA and RNA at the level of a single gene and of a whole genome.

5 Plan and complete experiments in molecular biology

6 Demonstrate skills in critical thinking and analysis

1.Command multiple skills and literacies to enable adaptable lifelong learning

2. Demonstrate knowledge of Indigenous Australia through cultural competency and professional capacity
3. Demonstrate comprehensive, coherent and connected knowledge
4. Apply knowledge through intellectual inquiry in professional or applied contexts
5. Bring knowledge to life through responsible engagement and appreciation of diversity in an evolving world

Fundamental Concepts and Methods in Molecular Biology

Created in 1983 by Kary Mullis, the polymerase chain reaction (PCR) is presently a typical and regularly key strategy utilized in medicinal and natural research labs for an assortment of utilization. PCR is a quick, economical and basic technique for replicating specific DNA molecules from very small quantities of the DNA material, even in cases where that source DNA is of poor quality. The technology does not use radioisotopes or dangerous synthetic substances making it safe and appropriate for numerous applications. One of the reasons for amplifying DNA is to make various duplicates of a piece of DNA which is extremely rare. For instance, a forensic researcher may need to amplify little pieces of DNA from a crime scene to facilitate investigations. Also, one may need to make comparisons between two or more DNA samples to ascertain which one is more abundant

The PCR

The chain reactions capitalize on the ability of an enzyme called polymerase to make new DNA strands complementary to the gene of interest. A polymerase will orchestrate a corresponding arrangement of bases to any single strand of DNA as long as it has a double-stranded beginning point. The DNA of interest forms the template strand in which the DNA polymerase adds nucleotides onto it at the 3’-OH group. Therefore, the DNA polymerase requires short single-stranded pieces of DNA called oligonucleotides complementary to the desired DNA or a section of it. These oligonucleotides are referred to as primers which with the activity of the enzyme amplify the specific regions of a DNA strand. In most of these reactions 0.1 to 10-kilo base pairs (kbp) of small pieces of DNA are amplified. However, some other techniques can achieve amplification of up to 40kbp (Tellinghusen and Spiess, 2014, pp.76-82). The available substrates influence the amount of the products because as the reaction progresses they get used up leading to product reduction (Carr and Moore, 2012, p.37640).

For a standard set-up, the PCR requires various components and chemicals. A template DNA containing a region for amplification together with a DNA polymerase enzyme that will amplify it is required. The enzyme ought to be heat resistant since the reaction uses extreme temperatures. Taq and Pfu polymerase are the most common catalysts used to drive these reactions (Hommelsheim et al., 2014, p.5052). The DNA needs two DNA primers complementary to the double DNA strands which form the anti-sense and sense strands. The primers have to be two for the activity of the DNA polymerase is only initiated when it binds to double strands. The DNA polymerase obtains nucleotides from deoxy-nucleoside triphosphates (dNTPs) to make the new strand. Therefore, they have to be available for the procedure. Also, a suitable chemical environment for the enzyme is required and this is provided by a buffer solution. Bivalent cations such as magnesium and monovalent cations for example potassium are necessary too for the reaction for they speed up the process of synthesizing the new genes.

The reactions are carried in a thermal cycler allows for heating and cooling at various stages of the PCR. Essentially, the reaction comprises of about 20-30 repetitive temperature changes referred to as cycles. Besides the heat variations, the time allowed for each reaction at every cycle depends on other parameters such as the concentration of the ions and dNTPs, temperature at which the primers melt as well as the type of the enzyme used (Xu et al., 2012, p.246). The stages for most PCR methods are initialization, denaturation, annealing, and elongation.

Gene Expression and Regulation

In the initiation stage, the DNA polymerase is activated by heat. The reaction chamber is heated to a temperature of 94-98 °C for a duration of 1-10 minutes. After the activation of the DNA polymerase follows the denaturation of the double-stranded template DNA. In this stage, the double-stranded DNA is separated into single strands by breaking the hydrogen bonds that exist between the complementary genes. A temperature of 94-98 °C is maintained for about 20-30 seconds leading to the formation of two single strands of DNA molecules (Svobodova et al., 2012, pp.835-842).

Annealing is the next step in which the temperatures are lowered to 50-65 °C for about 20-40 seconds to enable the primers to bind to the single-strand template DNA. The two primers bind to the single strands at the 3’ end of the target genes. The primers have single strands and very short, therefore they bind to only very short sequences on the target points of the template. Care is to be taken to provide the appropriate annealing temperatures for they affect the efficiency and specificity of the primers. In case the temperature is very high the primer may fail to bind and if very low the primer will not bind properly. The temperature should be reduced enough to allow hybridization and increased enough to allow for specificity in the hybridization (Zhang, Chen, and Yin, 2012, p.208). Typically, a temperature lower than the melting temperature by 3-5 °C of the primers used is appropriate for annealing to take place (Lorenz, 2012, p.63). The bonding is facilitated by the formation of stable hydrogen bonds between the complementary strands bases. These bonds cannot be formed unless the primer sequences closely match with the complementary strand base sequences. After successful binding of the primer, now the DNA polymerase binds to the hybrid, primer and template, and stars forming the new DNA.

In the elongation step the DNA polymerase synthesizes a new DNA strand that is complementary to the template DNA from the 5’ to 3’ end (Harrington et al., 2013, pp.1296-1303). This activity is effected through the addition of the free dNTPs in the reaction mixture. The temperatures at this step are determined by the polymerase used so as to optimize its activity. In case Taq polymerase is used, the temperature commonly used is 72 °C. However, the applicable range here is 72-78°C. The duration here is influenced by both the DNA polymerase utilized and the required length of the target DNA. However, by the rule of thumb most the polymerases amplify about one thousand base pairs in a minute under optimal conditions whereby at every extension the DNA sequences of the new strand increase by twofold. These new strands together with the original template form the DNA templates for each successive cycle bringing about an exponential amplification of the target genes.

The denaturation, annealing and extension reactions make one cycle. The number of cycles can be many depending on the amount of DNA required. Using the formula of 2ⁿ whereby n is the number of the cycles one can determine the number of copies of DNA formed at each cycle (Liu et al., 2015, p.71).

Comparing DNA Sequences and Analysis

The final elongation step follows although it is optional. This step takes place for 5-15 minutes at a temperature of 70-74°C to ensure all the single strands have been elongated. Finally, the reaction is cooled to a temperature of 4-15°C for unspecified duration serving as a temporal storage of the products. This step is called the final hold.

Products of these reactions can be checked to ascertain whether the reaction was successful using agarose gel electrophoresis. In this method, the products are run on the electrophoresis where they get separated on the basis of their sizes which are compared with a DNA ladder (Hopper, 2014, p.185).

The introduction of PCR technology in forensic science has made a breakthrough solving many challenges related to crime investigation. Majorly, this technology is utilized for DNA profiling which encompasses DNA typing, genetic fingerprinting and DNA testing. In this case, the forensics use DNA profiling to identify a criminal ruling out other suspects. The PCR methods in forensics focus on amplification of a variable number tandem repeats (VNTRs) which are similar for closely related individuals. Contemporary, forensics use PCR technology to amplify a class of VNTRs referred to short tandem repeats (STRs). This technique makes use of the short repeating sequences with 3-5 repetitive bases of the highly polymorphic DNA regions to discriminate unrelated people (Yang, Xie, and Yan, 2014, pp.190-197).

DNA samples from crime scenes are collected for example from cigarette butts providing pieces on DNA to be amplified even if they are very small. Then using particular sequence primers, the STR loci are amplified by PCR (Yang, Xie, and Yan, 2014, pp.190-197). The design of the primers enables the amplification of the STR loci from any individual. The copies of DNA obtained are then processed through electrophoresis whereby they are detected and separated forming unique patterns of bands. These bands form a genetic fingerprint whose unique pattern is utilized to match the suspect’s DNA that was found at the crime scene (Romos and Vallone, 2015, pp.90-99).

Conclusion

The breakthroughs exhibited in various fields that involve DNA in their research can be attributed to the PCR technology. Besides, the standard PCR there are many other PCRs such as the real-time PCR that has added advantages over the standard as it improves efficiency. The benefits the PCR technology has enhanced in science and other fields cannot be overestimated.  

References

Carr, A.C. and Moore, S.D., 2012. Robust quantification of polymerase chain reactions using global fitting. PLoS One, 7(5), p.e37640.

Harrington, C.T., Lin, E.I., Olson, M.T. and Eshleman, J.R., 2013. Fundamentals of pyrosequencing. Archives of Pathology and Laboratory Medicine, 137(9), pp.1296-1303.

Hommelsheim, C.M., Frantzeskakis, L., Huang, M. and Ülker, B., 2014. PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications. Scientific reports, 4, p.5052.

Hopper, W.R., AXXIN Pty Ltd, 2014. Nucleic acid amplification and detection apparatus and method. U.S. Patent Application 14/376,185.

Liu, M., Hu, P., Zhang, G., Zeng, Y., Yang, H., Fan, J., Jin, L., Liu, H., Deng, Y., Li, S. and Zeng, X., 2015. Copy number variation analysis by ligation-dependent PCR based on magnetic nanoparticles and chemiluminescence. Theranostics, 5(1), p.71.

Lorenz, T.C., 2012. Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. Journal of visualized experiments: JoVE, (63).

Romsos, E.L. and Vallone, P.M., 2015. Rapid PCR of STR markers: Applications to human identification. Forensic Science International: Genetics, 18, pp.90-99.

Svobodová, M., Pinto, A., Nadal, P.O.S.C.K. and O’Sullivan, C.K., 2012. Comparison of different methods for generation of single-stranded DNA for SELEX processes. Analytical and bioanalytical chemistry, 404(3), pp.835-842.

Tellinghuisen, J. and Spiess, A.N., 2014. Comparing real-time quantitative polymerase chain reaction analysis methods for precision, linearity, and accuracy of estimating amplification efficiency. Analytical Biochemistry, 449, pp.76-82.

Xu, G., Hu, L., Zhong, H., Wang, H., Yusa, S.I., Weiss, T.C., Romaniuk, P.J., Pickerill, S. and You, Q., 2012. Cross priming amplification: mechanism and optimization for isothermal DNA amplification. Scientific reports, 2, p.246.

Yang, Y., Xie, B. and Yan, J., 2014. Application of next-generation sequencing technology in forensic science. Genomics, proteomics & bioinformatics, 12(5), pp.190-197.

Zhang, D.Y., Chen, S.X. and Yin, P., 2012. Optimizing the specificity of nucleic acid hybridization. Nature chemistry, 4(3), p.208.