Direct Colony Sequencing

 

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).


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What is colony sequencing?

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.

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RCA Sequencing Data in 24 Hours**


Using RCA for DNA amplification expedites the Sanger sequencing process and allows your data to be delivered in as few as 24 hours** from sample receipt at Azenta.


GENEWIZ AAV-ITR Protocol

Rolling Circle Amplification Sequencing

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.

RCA Sequencing

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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.

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.


Direct Colony Sequencing Features & Benefits

Time and cost savings: no need to grow culture and prepare DNA
High-quality data: >600 bases Phred20 reads
Project design and consultation: highly-trained team of Sanger sequencing specialists available throughout the project 
Fast turnaround: receive your Sanger data in as fast as 24 hours** from sample receipt at Azenta
Online ordering: 24/7 online ordering and data retrieval system
GENEWIZ universal primers: select from a wide range of primers available at no additional cost
**Results in as fast as 24 hours for samples received at our Germany facility. Results in 36-48 hours for samples submitted to our UK facility.

RCA Sequencing 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.

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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 additional details on bacteria templates.

 

HOW TO ORDER


Email EU: sanger.europe@azenta.com | Phone: +49 (0)341 520 122-41

   Email UK: genomics.service.uk@azenta.com | Phone: +44 (0) 1279 873837

GENEWIZ from Azenta Life Sciences

European GENEWIZ Headquarters

GENEWIZ Germany GmbH, Bahnhofstrasse 86, 04158 Leipzig | +49 (0)341 520 122-41

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