Overview And Application Of Polymerase Chain Reaction (PCR)

In this assessment item you are asked to firstly provide a detailed overview of the theory, methodology and components of PCR. This will include details relating to the DNA polymerase that is used (heat stable, hot start polymerases, high-fidelity proof-reading DNA polymerases), the PCR reaction (understanding the steps that make up each cycle) and concepts such as annealing, denaturation, melting point, forward and reverse (or sense and antisense) primers, amplicons and amplification. The second part of the assessment will involve research into how PCR can be applied. You can choose an ex-ample from the fields of human (e.g., medical science or forensics), plant (e.g., transgenic plants) or animal (e.g., ecology and conservation) science and describe how PCR is applied in that context. You may describe the use of PCR in a research setting or look for examples where PCR is being used in an applied setting; for example, PCR is a critical part of many genetic tests, it is also part of DNA sequencing that is used to determine the DNA sequences of living and extinct organisms.

Theory, Methodology, and Components of PCR

The polymerase chain reaction is used to amplify DNA (or RNA). The technique was invented in 1985 by Kary B. Mullis (Smithsonian, 2004). He received the Nobel prize in 1993 for the invention. When DNA is available in minute amounts (in nano grams or micrograms) it is difficult to analyse it  through gel electrophoresis or employ it to other uses, the PCR is used to increase the amount of DNA through a series of DNA synthesis reactions in vitro. Another way of putting this is that the PCR has made it possible to create an enormous number of copies of a template of DNA. Initially, the enzyme used for DNA synthesis was the E. coli Klenow fragment of DNA polymerase I but the enzyme had to be replenished after each cycle due to its heat labile nature (Schochetman, et al., 1988). It was later replaced with Taq polymerase, a DNA polymerase sourced from the thermophilic bacterium Thermus aquaticus isolated from hot springs. This helped in automation of the PCR and it became possible to amplify DNA in short periods of time with the invention of the PCR thermocycler. Another source of polymerase enzyme is Pyrococcus furiosus, a thermophilic archaebacteria. The DNA polymerase sourced from this organism has high fidelity due to the 3'-5'exonuclease activity that helps it to proof read the DNA. The Pfu polymerase therefore, is better than Taq polymerase at making copies of DNA that are error free (Lundberg, et al., 1991).

The PCR thermocycler requires several additions before a series of reactions can form the numerous copies of DNA. The DNA template, that is the DNA to be amplified. Two single stranded DNA primers, that are complimentary to the ends of the template DNA. Taq polymerase to copy the DNA and nucleotides A, G, C and T that are used for incorporation into the newly synthesized strands of DNA. At times, an RNA template may be used, the enzyme reverse transcriptase is used to generate a DNA which is then amplified (Chakraborty & Kidd, 1991).

Denaturation

The first step is the denaturation of DNA for separation of strands. The step requires increase in temperature to 94⁰C. The high temperature is required, so that the double stranded DNA template melts or its strands separate due to dissociation of the hydrogen bonds between the nitrogen bases of the complimentary strands. The DNA could be from a medical, a forensic or an archaeological source, in fixed pathological specimens, human hair, sperm cells , buccal mouth washes and even ancient mummies (Eisenstein, 1990).  

Steps of PCR reaction: Denaturation, Annealing, Extension

Primer annealing

Once the strands separate, the temperature is lowered and due to presence of the primers in the mix, the primers anneal to the ends of the template DNA. Since there are two strands of DNA, a forward primer anneals with one strand and a reverse primer anneals with the other strand. The primers thus define the two ends of the stretch of DNA that is to be amplified. The primers are synthesized in vitro and added to the reaction mixture. Even if the target DNA sample is impure and less it can still be amplified (Eisenstein, 1990). It is important that the primers do not anneal to each other.

Extension

Once the primers bind the DNA polymerase can synthesize the DNA strand. If Taq polymerase is employed, it can extend the strand at 72⁰C. About 1000 bases can be added to DNA per minute in a thermocycler. The result of each round of amplification yields a greater number of DNA strands than the earlier and the number increases exponentially, such as, 1 amplifies to 2, then 4, 16, 256........, each product is called an amplicon (Sawyer, 2011).

(Yourgenome, 2016).

Depending on their properties DNA polymerases can be chosen for PCR based on the following properties:

Specificity

One of the major problems encountered during PCR is non-specific amplification of DNA. Misprimed targets or primer dimers may get amplified by the DNA polymerases during a PCR cycle. Setting up a PCR on ice reduces non-specific amplification. At times the addition of DNA polymerase is delayed until the annealing step of the first cycle. This technique is called ''hot start'' and it reduces non-specific amplification.

Thermostability

DNA polymerase employed for PCR should be able to withstand temperatures upto 90⁰C because the denaturation of DNA occurs at high temperature. Taq polymerase and Pfu polymerase are examples of thermostable polymerases.

High fidelity

When DNA polymerase has ability to proofread, it ensures accuracy of DNA replication. When the amplified DNA has to be used for sequencing or cloning, it is important that it be amplified with high fidelity and is free of errors in sequence. When an incorrect nucleotide is incorporated, there is a unfavorable base pairing kinetics, the polymerase stalls and the 3'-5' exonulease activity of the polymerase is able to excise the misincorporated nucleotide. Accuracy in DNA sequence is the outcome of high fidelity (Lodish, et al., 2000).

Processivity

The number of nucleotides that are added in a single event of polymerase binding is defined as the processivity of an enzyme. A polymerase with high processivity will synthesize DNA at a faster speed and has higher affinity for substrates. Processivity ensures that amplification of longer templates occurs more efficiently. Also, DNA with sequences that have a higher GC content and more secondary structures gets amplified better if processivity is high. Such polymerases re less affected by presence of inhibitors, such as, humic acid, heparin or xylan which may be found in blood or plant tissue (Thermofisher, n.d.). Polymerases with high processivity have a greater affinity for their substrates.

PCR concepts: Annealing, Denaturation, Melting Point, Forward and Reverse (or Sense and Antisense) Primers, Amplicons, and Amplification

Solving parental disputes, crimes where proof against a murder convict or rape convict is required.

Before the advent of PCR, electrophoretic techniques for proteins and DNA were utilised to differentiate between biological samples from different persons The use of polymorphic loci also known as very narrow tandem repeats or microsatellites that occur in the non-coding region of the genome and the number of repeats is unique to the DNA of each person through PCR is done to identify biological samples from crime or accident scenes. Most of these are deletion or insertion polymorphisms. The findings of DNA fingerprinting and PCR led to their use for forensic analysis (Gill, et al., 1985).

(Bitesizebio, n.d.)

Using PCR, the insertion or deletion polymorphisms can be compared rather quickly in two steps. Amplification and fractionation on the basis of size on a gel followed by ethidium bromide staining. The pattern of bands on the electrophoresis gel is unique to every individual. When the starting material is degraded DNA and sample size is also small, it is difficult to detect the amplified fragments by size fractionation alone. Besides, the DNA sample could contain bacterial or fungal DNA (Decorte & Cassiman, 1993). The sample to be amplified should contain at least one intact DNA with the two primer binding regions. Primer design is done such that the VNTRs from any individual can get amplified. The unique pattern of bands is the genetic fingerprint and can be used to match the suspect's DNA with that found at the crime scene (Jeffreys, et al., 1985). A corresponding fingerprint that belongs to the suspect being investigated has to be used to match with the one from the crime scene.

A southern blot can be followed by a labelled DNA probe hybridisation to visualise amplified alleles of VNTRs. At times  hapten can also be added to the amplification reaction mixture, such as, digoxigenin or boitin33. In such cases direct detection is possible after a Southern transfer through an avidin-alkaline phosphatase reaction. If the labelling is done with  fluorescent dye, its detection through an automatic laser detection system on a synthesizer makes the whole process automated. Radioactive isotopes have also been used for labelling. The automated system is highly sensitive and fragment size of the amplified product may also be directly uploaded onto a software and stored in databases for further reference.

In conventional PCR protocols, DNA is extracted and quantified before it is loaded on to a thermal cycler. But very low levels of DNA may often be lost during these procedures. Direct PCR amplification from forensic samples helps to circumvent these steps and helps in retention of DNA that might be lost during extraction and quantification. This reduces error and allows for faster turnaround using less financial resources when processing samples.

Other than providing evidence from a crime scene in the court room against a murder convict or a rape convict, a PCR can also help fix paternity disputes. Newer advances in PCR technology make use of robotic arms to micropipette reagents in a 96 well microtitre plate.

References

Bitesizebio, n.d. https://bitesizebio.com/19816/how-are-crimes-solved-by-pcr/. [Online]
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Chakraborty, R. & Kidd, K., 1991. The utility of DNA typing in forensic work. Science, 254(5039), pp. 1735-1739.

Decorte, R. & Cassiman, J.-J., 1993. Forensic medicine and the polymerase chain. Journal of medical genetics, Volume 30, pp. 625-633.

Eisenstein, B., 1990. The Polymerase Chain Reaction — A New Method of Using Molecular Genetics for Medical Diagnosis. New England Journal Medicine, Volume 322, pp. 178-183.

Gill, P., Jeffreys, A. & Werrett, D., 1985. Forensic applications of DNA 'fingerprints'.. Nature, Volume 318, pp. 577-9.

Jeffreys, A., Wilson, V. & Thein, S., 1985. Hypervariable 'minisatellite'' regions in human DNA.. Nature, Volume 314, pp. 67-73..

Lodish, H., Berk, A., Zipursky, S. & al., e., 2000. Molecular Cell Biology. 4 ed. s.l.:W H Freeman.

Lundberg, K. et al., 1991. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus.. Gene, 108(1), pp. 1-6..

Sawyer, E., 2011. pcr_a_revolutionary_invention. [Online]
Available at: https://www.nature.com/scitable/blog/bio2.0/pcr_a_revolutionary_invention
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Schochetman, G., Ou, C.-Y. & Jones, W., 1988. Polymerase chain reaction. The journal of infectious diseases, 158(6), pp. 1154-1157.

Smithsonian, I. A., 2004. videohistory_catalog9577.html. [Online]
Available at: https://siarchives.si.edu/research/videohistory_catalog9577.html
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Thermofisher, n.d. dna-polymerase-characteristics.html#Specificity. [Online]
Available at: https://www.thermofisher.com/in/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/dna-polymerase-characteristics.html#Specificity
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Yourgenome, 2016. what-is-pcr-polymerase-chain-reaction. [Online]
Available at: https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction
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