amplicons

An amplicon is a fragment of DNA or RNA that has been produced by amplification of a target region. In molecular biology, the term most commonly refers to the product of amplification by polymerase chain reaction (PCR). The region between the forward and reverse primers is amplified, yielding many copies of the same fragment. Amplicons can also refer to cDNA derived from RNA templates in RT-PCR, or products of whole genome amplification, but the latter usage is less specific to targeted studies.

Generation occurs through cycles of denaturation, primer annealing, and extension, during which a DNA polymerase copies the target region and the number of amplicon molecules increases exponentially. Amplicons can be produced in singleplex reactions (one target) or multiplex reactions (several targets) in a single mixture.

Applications are broad. They include diagnostic assays for pathogen detection and genetic testing, genotyping, cloning, and sequencing. Amplicon sequencing involves sequencing amplified products and is widely used for targeted sequencing panels, microbial community profiling (such as 16S rRNA gene sequencing), and variant discovery in specified regions.

Design considerations are important for reliable results. Amplicon length typically ranges from about 100 to 1000 base pairs, with many sequencing workflows preferring 200–500 bp. Primer design should maximize specificity, minimize primer-dimers and secondary structures, and account for GC content and potential SNPs in binding sites.

Limitations include amplification bias, preferential amplification, and polymerase errors, which can affect quantitative accuracy and variant detection. Proper controls and validation, along with size verification by electrophoresis or sequencing, help ensure product identity and quality.