Direct Colony Sequencing services utilize rolling circle amplification (RCA) to enable Sanger sequencing of bacterial clone or phage sample templates without the need for plasmid preparation. RCA generates DNA for sequencing by random hexamer priming of circular templates. GENEWIZ RCA protocols can handle sequencing projects of any size and are optimized to produce high quality reads from both bacterial colonies and glycerol stocks as well as phage plaques and supernatants. Direct Colony Sequencing is also extremely effective when low amounts of DNA starting material are available (e.g. low copy plasmids).
Colony sequencing is a method in which plasmid DNA (pDNA) is extracted and amplified directly from microbial colonies, allowing for sequencing data to be produced without the need for plasmid purification. In addition to saving time by eliminating plasmid preparation steps, another key benefit of colony sequencing is its flexibility with the starting sample. Any size plasmid, with a high or low copy plasmid template, can be sequenced with direct colony sequencing. Direct colony sequencing is commonly used for screening complimentary DNA (cDNA) libraries and site-directed mutagenesis.
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.
*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.