How RCA supercharges NGS library prep

In this Mol Bio Minutes mini-episode, Laurynas Alijošius shares how Rolling Circle Amplification (RCA) provides a reliable, high-yield approach for amplifying circular DNA prior to next-generation sequencing (NGS). This isothermal method avoids the need for thermal cycling and even bypasses the need for specific primers—making it ideal for challenging viral genomes, rare targets, or field samples.

Powered by the strand-displacing phi29 DNA polymerase, RCA amplifies DNA with impressive sensitivity and minimal GC bias. Laurynas breaks down the steps of multiple displacement amplification (MDA), explains why exonuclease-resistant primers are important, and explores how engineered polymerases like EquiPhi29™ DNA Polymerase dramatically improve yield and reduce reaction times. RCA products can be cleaned up and debranched to support a range of downstream workflows, including nanopore sequencing and transcriptomics.

From single-cell genomics to phage-based applications and in vitro expression systems, RCA is more than just a pre-NGS step; it’s a versatile tool with broad utility. Whether you're stabilizing viral RNA or tackling ultra-low-input samples, RCA and whole genome amplification offer new flexibility for today’s demanding sequencing workflows.

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For Research Use Only. Not for use in diagnostic procedures.