What is polymerase chain reaction?
If you’ve ever worked in a molecular biology lab, you’ve probably had a polymerase chain reaction (PCR). PCR is an in vitro method in which a small amount of DNA can be copied many times in a short period of time. The PCR was invented in the early 1980s by Kary B. Mullis, who later received the Nobel Prize in Chemistry for her work. Since then, PCR has become a standard and essential practice in molecular biology and can be used in a variety of scientific techniques, such as cloning or molecular diagnosis.
The beauty of PCR is that it can amplify DNA with just a short list of reagents and multiple heating and cooling steps. PCR relies on heat-resistant DNA polymerase from thermophilic bacteria Thermos aquaticus (Taq). Taq polymerase is therefore a heat-resistant enzyme that can withstand temperature changes. Taq was first identified in the late 1960s during a survey of hot springs in Yellowstone National Park.
In addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template DNA to be amplified, and single-stranded DNA primers that bind upstream (5′) and downstream (3′) to the DNA region of interest. Primers are essential for this process, as DNA polymerases require an existing strand of DNA to attack the nucleotides.
Using these reagents and a series of heating (denaturation) and cooling (annealing) steps, Taq polymerase can copy DNA between primers using dNTPs.
Let’s go into detail about the 3 basic steps of a PCR reaction:
- Denaturation – To amplify DNA, the two strands of template DNA must first be separated. This is done by heating the dsDNA template to a point where the hydrogen bonds between the base pairs break. This leads to the separation of the two strands of DNA.
- Annealing – The temperature is then reduced to a range where the forward and reverse primers are stable. At this temperature, primers can glow with single-stranded DNA strands. DNA polymerase is also stable at this temperature and can bind to primers.
- Extension – The temperature is then slightly increased to the ideal temperature for Taq polymerase (70-75oC). At this temperature, Taq polymerase can synthesize and elongate target DNA quickly and accurately.
Types of PCR
Since the invention of PCR, different PCR methods have been developed for different scientific applications. All of these PCR methods use the same setup and steps as the basic PCR but differ in how the PCR products are analyzed.
End point PCR
Endpoint PCR, as the name suggests, analyzes the final product of the PCR temperature cycle. The final PCR product is often visualized on a diagnostic agarose gel to confirm the presence, size, and relative amount of the product. Endpoint PCR is most commonly used in molecular cloning, sequencing, and genotyping. It is extremely affordable, but not as quantitative as other PCR methods. In theory, scientists should be able to determine the amount of DNA after a PCR reaction, since the amplicon doubles with each reaction cycle. However, it is common for dNTPs and other reagents to decay during later cycles, which can delay or stop PCR amplification. Other times the PCR reaction may not be 100% effective and produce a small amount of product.
Quantitative polymerase chain reaction, qPCR
Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, is a PCR technique used to measure an initial concentration of DNA using PCR. qPCR requires the addition of a probe-based fluorescent dye intertwined with each dsDNA and the use of a fluorometer function built into the thermal cycle to measure the fluorescent output. With this fluorescent dye, DNA concentration is continuously detected during PCR reaction cycles using a fluorescent signal. The signal increases in proportion to the amount of product produced in each cycle.
To determine the initial concentration of template DNA, the fluorescent signal during the reaction is compared to a standard curve of amplified DNA of known initial concentration. The cycle in which unknown DNA is detected against the standard curve can be used to determine the amount of starting material in the sample.
qPCR it can also be used to quantify RNA levels using qPCR reverse transcription (RT-qPCR). The first step in this process requires the RNA to be converted to cDNA using reverse transcription and the cDNA is then quantified by qPCR. qPCR is used in a variety of applications, including gene expression, the study of copy number variation, and molecular diagnostics.
Digital droplet PCR, ddPCR
Digital droplet PCR or ddPCR is a method that provides an ultrasound-sensitive absolute nucleotide concentration, unlike qPCR, where results may vary between replications. ddPCR can be used to quantify rare DNA sequences such as rare alleles or mutations. In addition, ddPCR does not require a reference or standard curve, which can be time-consuming and difficult to correct. ddPCR uses a water-oil emulsion droplet technology that splits the PCR reaction sample into approximately 20,000 droplets. Each drop contains the material needed for PCR amplification. After the PCR reactions, each drop is analyzed by a drop reader, which measures the fluorescence amplitude of each drop. The fraction of PCR positive fluorescent droplets is determined and then analyzed using Poisson statistics to determine the concentration of original template DNA in the sample.
Multiplex PCR, as the name suggests, is a method where multiple targets can be amplified in a single PCR experiment using multiple primers in a single PCR reaction. This is an extremely useful PCR method that can help you save time and effort in the lab.
There are two main categories of multiplex PCR:
Single template PCR reaction: A standard is amplified using multiple sets of forward and reverse primers.
Multiple Template PCR – Multiple templates with different primer pairs aligned with the target region of each template are used in one reaction.
Multiplex PCR is often used to identify diseases or pathogens. Scientists can simultaneously detect multiple pathogens in a sample, saving time and effort. Scientists can also use multiplex qPCR to quantify concentrations of different incipient DNA patterns in a sample. However, it is important to note that multiplex PCR is more complex to develop and generally less sensitive than PCRs using a single primer, as mentioned above. Multiplex PCR is also used in high-throughput SNP genotyping, gene analysis, and RNA detection.