What is Polymerase Chain Reaction?
What is PCR?
Polymerase chain reaction (PCR in short) is a core technique in molecular biology. The primary function is to duplicate millions (or even billions) of one DNA sequence.
This region of DNA can be anything of interest to the experimenter.
- Researchers can understand gene functions.
- Forensic scientists can match it with a suspect DNA sample.
Generally, the goal of PCR is to generate enough target DNA regions for analysis or other use.
For example, we can send PCR amplified DNA for sequencing. We can observe it by gel electrophoresis. Or even clone into a plasmid for further experiments.
To start, heat the sample to denature the DNA or separate it into two single-stranded DNA. Next, the "Taq polymerase" enzyme synthesizes and constructs two new DNA strands. The original strand is the template. The process leads to the duplication of the original DNA. Each new molecule contains an old and a new DNA strand. Then each of these chains can create two new copies, and so on. We repeat the denaturation and synthesis cycle up to 30 or 40 times. It will produce more than 1 billion exact copies of the original DNA fragment.
This reaction takes place in a small reaction tube (0.2-0.5 ml volume) with a thermal cycler (volume of 10-200 μL). Thermal cycler heats and cools the reaction tube to reach the required temperature. Many modern thermal cyclers apply the Peltier effect. It allows the block holding the PCR tube to be heated and cooled 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 need a coat of oil on the top of the reaction mixture or a wax ball in the tube.
How does PCR work?
Taq polymerase, primers, template DNA, and nucleotides are critical components. We assemble them in a tube together with the cofactors required by the enzyme. Then repeat heating and cooling cycles to synthesize DNA.
The basic steps are:
1. Denaturation (96 °C): The reaction is heated to separate or denature the DNA strands. It 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 copied DNA region. If the response is effective (works well), the target area can duplicate one or a few copies to billions.
That is because it is not just the original DNA used as a template each time. Conversely, new DNA made in one round can apply to a template for the next round of DNA synthesis. Many primer copies and Taq polymerase molecules are floating around in the reaction. So DNA molecules can approximately double in each cycle.
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
Seminar Topic: How does a COVID-19 PCR test work?
The COVID-19 PCR test has three key steps:
Step 1. sample collection
The sample is collected using a swab to collect respiratory material found in the patient nose. The cotton swab contains a soft tip on a long, flexible stick inserted into the nose. Nasopharyngeal and Nasal Swabs are the two common types. Any type of swab is enough to collect materials for COVID-19 PCR testing. After collection, seal the swab in a tube and send it to the laboratory.
Step 2. extraction
When laboratory technicians receive a sample, they perform an extraction process. It separates genetic material from the sample. Genetic material from any viruses present is also included.
Step 3. PCR
The PCR step uses special chemicals and a thermal cycler to cause a reaction. It will make 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 interpreted by medical software for a positive test result.
Seminar Topic: What do COVID-19 PCR test results mean?
A positive test result means that the patient is likely to have COVID-19. Most people have mild illnesses and can recover safely at home without medical care. If the symptoms worsen or the patient have questions or concerns, please contact the healthcare provider.
A negative test result infers a likelihood of not being infected with COVID-19 at the test time. The patient can be infected with SARS-CoV-2, but there is not enough virus in the body detected in the test.
For example, the patient is infected, but without symptoms, this may happen. In another case, the patient can be infected with COVID-19 for more than a week before being tested.
So, a negative test does not guarantee safety at all times. After the test, the patient may be exposed to COVID-19, be infected and spread the SARS-Cov-2 virus to others.
If the test is positive, talk to the healthcare provider, stay home and separate oneself from others. If a negative test, please continue to take steps to protect self and others from COVID-19.
COVID-19 and PCR Testing, Cleveland Clinic
Polymerase Chain Reaction (PCR), by NIH Institute
Polymerase chain reaction (PCR), Khan Academy
Polymerase chain reaction, Wikipedia