Direct Colony Sequencing

GENEWIZ's Direct Colony Sequencing services utilizes rolling circle amplification (RCA) to enable the Sanger sequencing of bacterial colonies 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. Direct Colony Sequencing is also extremely effective when low amounts of DNA starting material are available (e.g. low copy plasmids).

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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.

The RCA Process

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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.
Polymerase is utilized due to its high stability, processivity, and high fidelity. This allows prolonged strand displacement over multiple rounds of amplification.
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.
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.


 

FEATURES & BENEFITS

Time savings and cost savings - no need to grow culture and prepare DNA
>600 bases of Phred20 reads
Free GENEWIZ Universal Primers
Project design and consultation
Analysis using ABI Prism 3730xl DNA analyzers
Fast turnaround
GENEWIZ Online Ordering and Data Retrieval System - 24/7 web-based
Customized troubleshooting

APPLICATIONS

shRNA cloning and library screening; Eliminate plasmid preps and sequence colonies directly.

DNA methylation analysis: Save time & obtain high-quality reads by cloning PCR products directly and sequencing the bacterial colonies.

SAMPLE SUBMISSION OPTIONS

Sample Submission Options

Glycerol Stock* Send 96-well plates; samples must be shipped on dry ice. Please label with media type.
Bacterial Colonies* 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.

 

HOW TO ORDER

*Samples must arrive at the GENEWIZ New Jersey laboratory before 10:00 am EST to qualify for Same Day service.  Note that direct-sequencing templates are not available for our Same Day service.



Email: Sanger.Europe@genewiz.com | Phone:  +49 (0) 341 520 122 - 41