What is Polymerase Chain Reaction?
What is PCR?
Polymerase chain reaction (PCR) is an regular laboratory method applied to make several (millions or billions!) of details of DNA. This region of DNA can be anything of to the experimenter. It might be a gene for which researchers desire to recognize its function, or a genetic marker made use of by forensic researchers to match crime scene DNA with a suspect.
Typically, the objective of PCR is to produce sufficient target DNA regions for evaluation or various other usage. For example, DNA magnified by PCR can be sent for sequencing, observed by gel electrophoresis, or cloned right into a plasmid for further experiments.
Applications of the technique consist of DNA cloning for sequencing, genetics cloning and also , gene mutagenesis; building of DNA-based phylogenies, or functional evaluation of genes; diagnosis and monitoring of congenital diseases; amplification of old DNA; evaluation of fingerprints for DNA profiling (for instance, in forensic scientific research as well as parentage screening); as well as discovery of microorganisms in nucleic acid tests for the medical diagnosis of transmittable diseases.
To use PCR to amplify DNA fragments, first heat the sample to denature the DNA or separate it into two single-stranded DNA. Next, an enzyme called "Taq polymerase" synthesizes-constructs-two new DNA strands, using the original strand as a template. This process leads to the duplication of the original DNA, and each new molecule contains an old and a new DNA strand. Then each of these chains can be used to create two new copies, and so on. The cycle of denaturation and synthesis of new DNA is repeated up to 30 or 40 times, resulting in more than 1 billion exact copies of the original DNA fragment.
This reaction is usually carried out in a small reaction tube (0.2-0.5 mL volume) of a thermal cycler in a volume of 10-200 μL. The thermal cycler heats and cools the reaction tube to reach the temperature required for each reaction step (see below). Many modern thermal cyclers utilize the Peltier effect, which allows the block holding the PCR tube to be heated and cooled simply by reversing the current. The thin-walled reaction tube allows good thermal conductivity to achieve rapid thermal equilibrium. Most thermal cyclers have a heated lid to prevent condensation on the top of the reaction tube. Old thermal cyclers without a heating cover require a coat of oil on the top of the reaction mixture or a wax ball in the tube.
How does PCR work?
The key components of a PCR reaction are Taq polymerase, primers, template DNA and nucleotides (DNA building blocks). These components are assembled in a tube together with the cofactors required by the enzyme and undergo repeated heating and cooling cycles to synthesize DNA.
The basic steps are:
1. Denaturation (96 °C): The reaction is heated strongly to separate or denature the DNA strands. This provides a single-stranded template for the next step.
2. Annealing (55°C-65°C): Cool the reaction so that the primer can bind to the complementary sequence on the single-stranded template DNA.
3. Extension (72 °C): Increase the reaction temperature to allow Taq polymerase to extend the primer to synthesize a new DNA strand.
This cycle is repeated 25-35 times in a typical PCR reaction and usually takes 2-4 hours, depending on the length of the DNA region being copied. If the response is effective (works well), the target area can be increased from one or a few copies to billions.
That's because it's not just the original DNA that is used as a template each time. Conversely, new DNA made in one round can be used as a template for the next round of DNA synthesis. There are many primer copies and many Taq polymerase molecules floating around in the reaction, so the number of DNA molecules can approximately double in each cycle. This exponential growth model is shown in the figure below.
What is PCR used for?
To name a few at high-level:
- Selective DNA isolation
- Amplification and quantification of DNA
- Medical and diagnostic applications
- Infectious disease applications
- Forensic applications
- Research applications
Special Topic: How does a COVID-19 PCR test work?
The COVID-19 PCR test has three key steps:
Step 1. sample collection
The sample collection is done using a swab to collect respiratory material found in your nose. The cotton swab contains a soft tip on a long, flexible stick that is inserted into your nose. There are different types of nasal swabs, including nasal swabs that immediately collect a sample in the nostril and nasopharyngeal swabs that are collected deep into the nasal cavity. Any type of swab is sufficient to collect materials for COVID-19 PCR testing. After collection, the swab is sealed in a tube and sent to the laboratory.
Step 2. extraction
When laboratory technicians receive a sample, they perform a process called extraction, which separates genetic material from the sample, including genetic material from any viruses that may be present.
Step 3. PCR
Then, the PCR step uses special chemicals and a PCR machine called a thermal cycler to cause a reaction to take place, making millions of copies of a small part of the genetic material of the SARS-CoV-2 virus. During this process, if SARS-CoV-2 is present in the sample, one of the chemicals will fluoresce. This fluorescence is the "signal" detected by the PCR machine, and special software is used to interpret the signal as a positive test result.
Special Topic: What do COVID-19 PCR test results mean?
A positive test result means that you are likely to have COVID-19. Most people have mild illnesses and can recover safely at home without medical care. If your symptoms worsen or you have questions or concerns, please contact your healthcare provider.
A negative test result means that you may not have been infected with COVID-19 at the time of the test. However, it is possible to be infected with SARS-CoV-2, but there is not enough virus in your body to be detected by the test. For example, if you were recently infected but you have no symptoms, this may happen; or if you have been infected with COVID-19 for more than a week before being tested. Remember, a negative test does not mean you are safe at any time. After the test, you may be exposed to COVID-19, be infected and spread the SARS-Cov-2 virus to others.
If you test positive, talk to your healthcare provider, stay home and separate yourself from others. If your test result is negative, please continue to take steps to protect yourself and others from COVID-19.
Polymerase chain reaction (PCR) by Khan Academy
Polymerase Chain Reaction (PCR) Fact Sheet by NIH Institute
Polymerase chain reaction by Wikipedia
Polymerase Chain Reaction (PCR) by ThermoFisherCOVID-19 and PCR Testing by Cleveland Clinic