Amplicons are specific segments of DNA or RNA extensively copied in a laboratory. They are like millions of identical copies of a selected genetic “page” from an organism’s genome. This process is fundamental in molecular biology.
The Creation of Amplicons
Generating amplicons relies on Polymerase Chain Reaction (PCR). This method begins with a target DNA sample. The reaction mixture includes short synthetic DNA strands called primers, which define the start and end points of the region to be copied.
DNA polymerase, an enzyme that builds new DNA strands, is another component. Deoxynucleotides (dNTPs), the individual building blocks of DNA, are also required. The PCR process cycles through three main temperature-dependent steps.
First, denaturation involves heating the mixture to a high temperature to separate double-stranded target DNA into two single strands, breaking hydrogen bonds. Then, the temperature is lowered for annealing, allowing primers to bind to complementary sequences on the single-stranded DNA templates.
Finally, during extension, the temperature is raised to an optimal range for DNA polymerase. The polymerase synthesizes new DNA strands by adding dNTPs to the primers, extending them along the template. These steps repeat for many cycles, doubling the targeted DNA fragment and leading to an exponential increase in amplicon copies.
Analyzing Amplicons
Once amplicons are created, scientists use various techniques to analyze them. Gel electrophoresis is a common method that sorts DNA fragments by size. Amplicons are loaded into a gel matrix, and an electric current causes the negatively charged DNA to migrate towards the positive electrode.
Smaller fragments move more quickly through the gel, separating into distinct bands. By comparing the amplicon band’s position to a ladder of known DNA sizes, researchers verify if the correct DNA length was amplified. A single, appropriately sized band indicates successful amplification; multiple bands suggest unintended products.
Beyond size verification, amplicon sequencing reads the exact genetic code of copied fragments. This technique determines the precise order of nucleotides (A, T, C, G) within the amplicon. This detailed sequence information allows scientists to identify genetic variations, mutations, or specific genetic markers in the original DNA sample.
Practical Applications of Amplicons
The ability to create and analyze millions of copies of specific DNA or RNA regions makes amplicons useful across many scientific fields. In medical diagnostics, amplicons detect pathogens. For example, during the COVID-19 pandemic, amplicon-based PCR tests identified SARS-CoV-2 genetic material in patient samples, allowing rapid and sensitive virus detection. This approach is effective even when genetic material is present in very small quantities.
Amplicons also play a significant role in genetic research. They enable scientists to identify specific gene mutations associated with various diseases. By amplifying a gene region, researchers can sequence it to pinpoint genetic variants that might predispose individuals to conditions or influence disease progression.
In forensics, amplicons are used for creating DNA profiles from minute or degraded biological samples found at crime scenes. Even a tiny trace of blood, hair, or skin cells can yield enough target DNA for amplification, producing sufficient quantities of specific genetic markers for identification.
Amplicons are also employed in environmental science to identify and characterize microbial species in diverse samples like water or soil. By targeting conserved genetic regions, scientists can amplify and sequence them to determine the types and abundance of microorganisms in an ecosystem.
Amplicons Versus Genes
It is helpful to distinguish between an amplicon and a gene. A gene is a naturally occurring functional unit of heredity within an organism’s genome. Genes contain instructions for building proteins or functional RNA molecules and are fundamental units of biological inheritance.
In contrast, an amplicon is a laboratory-produced copy of a specific, targeted DNA or RNA region, generated artificially through processes like PCR. The copied region can be an entire gene, a small segment, or a non-coding region useful for identification or analysis.