Rolling circle amplification (RCA)
Based on the natural rolling circle replication from bacterial plasmids, bacteriophage, and viruses, rolling circle amplification (RCA) technique was developed and widely used in molecular biology research applications including preparation of the DNA template for Sanger sequencing, and biomedicine applications including cancer gene mutation detection and diagnostics.
RCA is an isothermal amplification method that provides benefits of easy use in point-care that the most popular PCR technique has limits in due to the same temperature or room temperature used in RCA. In addition, the RCA method produces large amount of amplified DNA with much lower error rate compared to routine PCR reactions due to the most common use of high fidelity phi29DNA polymerase.
phi29 DNA polymerase
phi29 is a DNA polymerase discovered in Bacillus subtilis bacteriophage phi29 by Dr. Margarita Salas in 1960s, who won the lifetime achievement award from the European Patent Office in 2019 and passed away later in the same year. Compared with other DNA polymerases found, the phi29 DNA polymerase has the following outstanding features that make it the great choice for RCA:
- Long processivity (synthesizing long DNA pieces without dissociating from the template) of >70,000 bp
- Low-error rate (mutations in 3X10-6 to 10-7 per base compared with Taq DNA polymerase at 1×10-3 per base)
- High yield of DNA amplification (generation of more than 10-50 µg of DNA from nanograms amounts of DNA within a few hours)
phi29 DNA polymerase applications in either RCA or whole genome amplifications
phi29 DNA polymerase plays an important role in modern genomics either through RCA or whole genome amplification (WGA). For achieving sufficient testing materials, phi29 DNA polymerase is used in Sanger sequencing, Next Generation Sequencing (NGS), and single nucleotide polymorphism (SNP) detection in microarray. The enzyme has also been widely used in diagnostic applications such as virus detection in clinical samples, whole genomics analysis of non-cultivable viruses, and more recent COVID-19 infection detection.
RCA used for plasmid amplification for Sanger sequencing
Due to great benefit of RCA using phi29 DNA polymerase, the method was used for preparation of Sanger sequencing templates from single colonies or minute amounts of liquid culture. Such a practice saves time from growth of bacterial culture overnight and isolation of plasmids. In this process, the single colonies containing different gene mutation sequences are taken and boiled first. The supernatant containing the plasmid is directly used for RCA. With generic primer hexamers, the phi29 DNA polymerase amplifies the plasmid template within a few hours at 30oC and the resulting amplified product can be directly sequenced by Sanger sequencing.
We provide two platforms of phi29 DNA polymerase products: the phi29 DNA polymerase itself and the ready-to-use RCA DNA amplification kit. You can try out these high-quality, cost-effective products yourself.