Polymerase chain reaction (PCR) method
Polymerase chain reaction (PCR) is a powerful laboratory technique used to amplify specific DNA sequences, enabling researchers to produce millions of copies from a minute DNA sample. The PCR process involves three essential steps: denaturation, where high temperatures separate the DNA strands; annealing, where short DNA sequences called primers bind to the template DNA; and extension, where a specialized enzyme, DNA polymerase, synthesizes new DNA strands. This cycle is typically repeated 20 to 25 times, leading to an exponential increase in the target DNA copies.
Invented by Kary Mullis in 1983, PCR has revolutionized fields such as forensic science, medical diagnostics, and biotechnology. It overcame previous challenges in DNA research, such as limited sample availability and difficulty in isolating target sequences. PCR's impact is profound; it has been instrumental in diagnosing diseases like HIV and tuberculosis, and it has adaptations like quantitative PCR (qPCR) to assess gene expression levels. The simplicity and effectiveness of PCR have made it a foundational tool in genetic research and clinical applications.
Polymerase chain reaction (PCR) method
Definition
Polymerase chain reaction (PCR) is a rapid technique that enables the copying of desired deoxyribonucleic acid (DNA) molecules. This technique consists of three steps: denaturation, annealing, and extension, repeated in a cyclical fashion.
In step one of PCR, the reaction is exposed to temperatures as high as 194° to 201° Fahrenheit (90° to 94° Celsius). The high temperature helps to separate the two strands of the DNA template by breaking the hydrogen bonds that hold the two strands together. This melting of the DNA duplex is called denaturation.
The second step in PCR is called annealing or hybridization and requires short single-stranded DNA sequences called primers. The reaction uses two primers, one complementary to each strand of the original DNA duplex. The primers bind to their complementary sequences (or anneals) on the template DNA and provide the free 3' OH (hydroxyl) group that DNA polymerase needs to copy the DNA. This sets the stage for the last step in PCR, extension. During extension, the DNA polymerase uses deoxyribonucleotides (dNTPs) to build the complementary strand on the template DNA.
The foregoing set of three processes (making up a cycle) is typically repeated several times, about twenty to twenty-five cycles, to allow for an exponential increase in copies of the target gene. A simple formula can be used to calculate the yield (number of copies of the DNA or gene template). According to this formula, the number of gene copies after n cycles of PCR is 2 n. For example, if a person starts with a single copy of the gene, the yield will be 225 at the end of a PCR reaction with twenty-five cycles.
Background
PCR was discovered by Kary Mullis (1944–2019) in 1983 while trying to develop a method that would allow the sequencing of single nucleotide polymorphisms (SNPs). SNPs are variations produced in DNA by alterations to a single nucleotide and, therefore, serve as the absolute genetic marker. Specifically, Mullis was trying to devise a rapid clinical assay for genetic disorders such as sickle cell disease that are caused by a single nucleotide polymorphism. DNA research in the 1980s faced two major challenges: not enough DNA and no easy way to separate a gene’s DNA from the genomic DNA (1 milligram of DNA contains about 200,000 copies of a person’s target gene). PCR offered a solution to both of these problems; one could amplify DNA rapidly and could obtain as many copies as needed of the target gene alone. For this discovery, Mullis was a cowinner of the Nobel Prize in 1993.
The PCR Reaction
A PCR reaction, which is typically around 50 milliliters, requires the following: DNA template or target gene (10 femtograms to 10 milligrams), primers (0.1-1.0 millimoles each), dNTPs (200 millimoles each), DNA polymerase (0.2-2 units) and Tris buffer (pH 8.0). Also, a PCR reaction requires MgCl2 (magnesium chloride), which is a cofactor for DNA polymerase. The DNA polymerase used in PCR is unique in that it can withstand high temperatures such as those used for melting DNA during the denaturation step. Before thermostable polymerases such as Taq and Vent were discovered, fresh polymerase had to be added after each round of PCR.
DNA in the cell is typically bound to packaging proteins such as histones. For a successful PCR reaction, the DNA template must be free of any inhibitory molecule (such as these packaging proteins) and thus easily accessible to the primers and polymerase. Therefore, once the DNA has been extracted from the cell, it is typically purified using some kind of enzymatic, mechanical, or chemical purification technique.
Next in the PCR process is the design of primer (forward and reverse primers). PCR primers are chemically synthesized nucleotide segments called oligonucleotides (or oligos) and are typically 18 to 30 bases long. Because primers are short sequences, misalignment can occur, leading to erroneous amplification. To minimize nonspecific binding, primers are preferably 18 to 21 bases long. The length of the PCR product is determined by the distance between the annealing sites of the two primers. Also important in primer design are factors such as guanine-cytosine content, polypurine or polypyrimidine stretches (multiples of any one nucleotide), the secondary structure of the primer (mutually complementary sequences within the primer can form “hairpin” structures), and the probability of a complementary sequence on the DNA residing anywhere except on the intended target gene (or region of amplification).
Once the PCR reaction is completed, the next important step is analysis of the PCR products with electrophoresis and various DNA intercalating dyes; this is followed by sequencing the amplimer (the amplified PCR product).
Impact
It is widely believed that since the discovery of the DNA double helix in the 1950s, few discoveries have transformed the field of biology as rapidly as PCR. This technique that allows the copying of millions of desired DNA beginning with minute DNA samples has accelerated progress in forensics and in medical diagnostics and has revolutionized biotechnology and biomedical research. For example, PCR has been used to improve the diagnosis and detection of HIV, tuberculosis, whooping cough, and, perhaps most well known in the 2020s, for detection of the SARS-CoV-2 virus.
Several modifications of the original PCR technique have been developed to meet the diverse needs of biomedical research. One such modification, real-time PCR or quantitative PCR (qPCR), is frequently used in drug discovery to look for and assess new putative drug target sites in humans. The qPCR method is also used to determine the number of working copies of a specific gene, and this measure of gene expression is important in analysis of genetic disorders. For example, in STEP (single target expression profiling), qPCR is used to compare active gene numbers in affected versus healthy (unaffected) persons, allowing for the study of the progression or development of genetic disorders.
Bibliography
Artika, I Made, et al. "Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis." Genes, vol. 13, no. 12, 2022, p. 2387, oi.org/10.3390/genes13122387. Accessed 17 Oct. 2024.
"Kerry Mullis." Britannica, 4 Sept. 2024, www.britannica.com/biography/Kary-Mullis. Accessed 17 Oct. 2024.
McPherson, Mike, and Simon Møller. PCR, 2nd ed. Taylor & Francis, 2006, doi.org/10.1201/9780429258381. Accessed 17 Oct. 2024.
Rabinow, Paul. Making PCR: A Story of Biotechnology. University of Chicago Press, 1997.
Van Pelt-Verkuil, E., et al., Principles and Technical Aspects of PCR Amplification. Springer, 2010.