Divergent primers are a specialized type of primer pair used in molecular biology techniques, primarily for amplifying specific genetic sequences.
Understanding Divergent Primers
According to research [5], divergent primers are defined as primers that cross each other back in primer pairs and reverse splicing sites. This description highlights their unique orientation compared to standard primers used for linear DNA or RNA.
Key Characteristics
- Paired Use: They are used in pairs, much like standard PCR primers.
- Orientation: Unlike primers that point outwards along a linear template, divergent primers are oriented to point towards each other when placed on a circular template.
- Target Site: They specifically target regions containing reverse splicing sites on circular transcripts.
Application: Amplifying Circular Transcripts
The primary use of divergent primers is to amplify specific circular transcripts [5]. Circular RNAs (circRNAs) are formed through a process called back-splicing, where a downstream splice donor joins an upstream splice acceptor. This creates a circular molecule.
Standard PCR primers designed for linear sequences wouldn't effectively amplify the junction formed by this back-splicing event. However, divergent primers are designed to sit across this junction, pointing inwards towards each other on the circular molecule. When amplification occurs, they specifically amplify the sequence spanning the reverse splicing site, confirming the presence of the circular form.
Why Use Divergent Primers for circRNAs?
- Specificity: They are crucial for specifically detecting and amplifying circular RNA isoforms, differentiating them from linear counterparts.
- Validation: Amplification using divergent primers and subsequent sequencing across the junction provides strong evidence for the existence of a circular transcript.
In essence, divergent primers are molecular tools specifically designed to leverage the unique structure of circular RNA molecules, allowing researchers to study these important non-coding transcripts.
