Sequencing of Bacteria by Rolling Circle Amplification (RCA)
RCA was identified in natural systems as a DNA replication mechanism frequently employed in plasmid and viral propagation. When random hexamers are coupled with a strand displacing DNA polymerase, many concatenated template copies are generated with no need for thermocycling (Dean et al., 2001). The synthesized strands are displaced after one round of RCA and are free for additional rounds of primer binding. Exponential amplification is achievable if the RCA reaction contains primers complementary to both template strands.
After lysis, bacterial samples are combined with dNTPs, polymerase, and random hexamers. The hexamers anneal to the circular template, and replication initiates at many sites simultaneously.
As the newly synthesized strands are displaced, a cascade of priming events facilitates exponential amplification of the template.
Upon completion the sample is ready for Sanger sequencing.
Note that RCA adds an additional 24 hours to the sequencing turnaround time.
Dean, Frank B. et al. “Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification.” Genome Research 11.6 (2001): 1095–1099.
SAMPLE SUBMISSION OPTIONS
Sample Submission Options
||Send 96-well plates; samples must be shipped on dry ice. Please label with media type.
||Array colonies in a grid on the agar plate and circle the colonies requiring sequencing. Numerically label each colony. Note that unnumbered plates will be processed as random pick. If using liquid culture, submit samples in 96-well agar stab plate plate with strip caps. Wrap agar plates in parafilm, and provide a cushion to protect against potential damage.
*Please note: RCA protocol is intended for circular templates <10 kb; linear templates will not be amplified sufficiently for direct Sanger sequencing.
Longer circular templates, or circular templates carried in bacterial strains with functional endA may not be reliably amplified, contributing to variable results
Please visit our Sample Submission Guidelines for bacteria and phage templates for additional details.