Effective research and advancements in life science would not be possible without the discovery of PCR. PCR has contributed greatly to innovations in biopharmaceuticals, biotech crops, and industrial biotechnology. The development of hundreds of technologies in the aforementioned areas, such as genetic cloning, forensics, and genetic analysis, is the result of PCR.
History of PCR
The idea to reproduce or make copies of a DNA segment (or DNA replication) in a laboratory tube (which normally takes place in a cell) was introduced by Kary Mullis in 1983. The invention eventually granted the scientist the 1993 Nobel prize in chemistry (shared with the Cetus Corporation) and brought on a revolution in research areas of biological sciences.
While Taq DNA polymerase had already been discovered at the time, its impact on molecular biology was not fully recognized without the introduction of PCR. PCR enabled scientists to amplify any DNA sequence in just a few hours using automated equipment rather than manual methods of DNA amplification.
What is PCR?
Polymerase chain reaction (PCR) is a rapid and inexpensive in vitro technique used to amplify copies of small segments of DNA. This process can produce billions of copies quickly and efficiently. It is also sometimes called “molecular photocopying”.
PCR has several applications in the area of medical and molecular biology. Some of them are given below:
- Selective DNA Isolation: PCR is used in labs to isolate specific DNA fragments by amplifying only a selected region of DNA. It can synthesize large amounts of pure DNA by using a small DNA sample. PCR also assists in generating hybridization probes required for southern or northern blots and DNA cloning.
- Medical and Diagnostic uses: PCR is used for diagnosing disease-associated genetic mutations, identifying infectious agents, and prenatal genetic testing. It can identify any chromosomal abnormalities or genetic mutations in a fetus, test parents for being genetic carriers of any diseases, and be used as a preimplantation genetic diagnosis tool to screen embryos for in vitro fertilization (IVF) procedures.
PCR assays are also used in the detection, diagnostic analysis, and sequencing of viral genomes. For example, a variant of PCR (RT-PCR) was extensively used to test COVID-19 patients infected with the SARS-CoV-2 viral genome.
Traditionally, these tests relied on the presence of antibodies that took days to appear in the bloodstream. The discovery of RT-PCR (real-time PCR) has facilitated the detection of even the smallest amount of viral genome in a host in just a matter of a few hours.
In RT-PCR, the enzyme reverse transcriptase uses RNA as a template and reverse transcribes RNA into DNA molecules, which is then amplified using PCR. Other than RT-PCR, some other types of PCR include:
- Hot-start PCR
- High fidelity PCR
- Multiplex PCR
- Microfluidic PCR
- Genetic research: PCR is used to study gene expression, evaluate the presence or absence of gene transcripts, manipulate genetic sequences, enrich sequencing samples, and genotyping. It was also extensively involved in the Human Genome Project (HGP) for most of the mapping procedures.
- DNA Fingerprinting: PCR is a benchmark technology in paternity testing and forensic investigations to find the source of DNA samples.
- Food Safety: PCR techniques can be used to detect pathogens in food and water and ensure the safety of eatables.
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How Does a Polymerase Chain Reaction Work?
A thermal cycler (or thermocycler) is used to run PCR reactions and amplify DNA sequences in vitro. It mimics the process of DNA replication that occurs inside a cell. A tube containing a reaction mixture of PCR reagents is put into the machine, which changes the temperature to suit each stage of the process. An essential requirement before performing PCR is the knowledge of sequence information of the target DNA.
The PCR reaction is completed in a few hours and requires five key reagents:
- DNA template: The sample DNA that contains the target sequence that needs to be amplified. The source of a DNA template can either be genomic DNA (gDNA), cDNA, or plasmid DNA.
- DNA Polymerase: PCR requires DNA polymerases that can work at high temperatures. The Taq polymerase is the most commonly used polymerase in PCR. It’s a thermostable DNA polymerase isolated from the bacterium Thermus aquaticus found in hot springs.
- Primers: Primers are short pieces of single-stranded DNA (approximately 15-30 base pairs), called oligonucleotides. It initiates the formation of new strands of DNA.
- Deoxynucleotide triphosphate (dNTPs): These are the four DNA nucleotides that serve as building blocks to synthesize new DNA strands.
- PCR Buffer: The main components for a PCR buffer are magnesium chloride (MgCl2), tris-HCl, and potassium chloride (KCl). They are required to maintain the optimal conditions throughout the PCR reaction.
After an initial denaturation step, where the DNA is heated to 95 for a few minutes (up to 10 depending on complexity), the three steps below are repeated cyclically.
At this stage, the templated DNA is heated up to 95°C for up to a minute. It produces two single strands of DNA by breaking the hydrogen bonds between DNA helices. This process is called nucleic acid denaturation.
In this stage, the reaction mixture is allowed to cool for 30 seconds to 1 minute. Then, primers bind or anneal to each single-strand DNA. The annealing temperature falls between 50 – 65°C. The exact temperature is 5°C below the melting temperature of the primers and depends on the length and sequence of primers used in the PCR reaction.
At this stage, the temperature is raised to approximately 72°C-74°C, the optimal temperature for Taq Polymerase, for about 1-2 minutes. The polymerase binds to the end of the primers and initiates the DNA synthesis using dNTPs. This leads to the formation of new strands of DNA.
At the end of one cycle, four single-stranded DNA molecules will be present in the PCR tube. Repeating the cycle over 20-30 times results in billions of copies of target DNA sequences.
One can then analyze the PCR product using agarose gel electrophoresis and check for the successful amplification of the target sequences.
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High-Quality Lab Solutions with Excedr
The discovery of PCR has revolutionized research in biotechnology, pharma, biomedical, and other life science areas. Today, it’s a common tool used in research labs with applications in detecting pathogens, forensic studies, genetic abnormalities, and food safety.
Thermal cyclers use the principle of PCR to amplify DNA samples in millions and billions of copies. It adjusts the optimal temperature at each stage of amplification that involves denaturation, annealing, and elongation of DNA.
A high-tech PCR thermocycler can cost as much as $25,000. Additionally, its high maintenance and cost of repairs make buying PCR equipment outright impractical for small biotech labs.
With Excedr, you can overcome the financial hurdles of equipment procurement and fully outfit your lab at an affordable cost without compromising quality. Here, you can procure the cutting-edge analytical, life science/biotech, or clinical equipment you need to operate at the highest level.