Direct Colony Sequencing

 

GENEWIZ's Direct Colony Sequencing services utilizes rolling circle amplification (RCA) to enable the 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).


RECEIVE YOUR SANGER SEQUENCING DATA IN JUST 24 HOURS**


Using RCA for DNA amplification expedites the Sanger sequencing process and allows your data to be delivered within 24 hours** of sample receipt at GENEWIZ.


GENEWIZ AAV-ITR Protocol

Sequencing of Bacteria & Phage 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

  • Building


    1
  • arrow
  • Building


    2
  • arrow
  • Building


    3
  • arrow
  • Building


    4
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.


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 and cost savings: no need to grow culture and prepare DNA
High-quality data: >600 bases of Phred20 reads
Project design and consultation: Ph.D.-level customer support throughout the project
Fast turnaround: receive your Sanger data within 24 hours** of sample receipt at GENEWIZ
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
**24-hour turnaround time guaranteed for maximum of 96 samples; excludes phage samples

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.

Antibody phage display library validation: Direct phage sequencing for faster screening and validation.

SAMPLE SUBMISSION OPTIONS

Sample Submission Options

Glycerol Stock* Send 1.5 ml tubes or 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 plate with strip caps. Wrap agar plates in parafilm, and provide a cushion to protect against potential damage.
Phage Supernatant* Please submit 50 ul of phage supernatant in 8-strip PCR tubes or 1.5 ml microcentrifuge tubes clearly labeled with sample name.
Phage Plaque Circle and number the plaques you submit for sequencing. For transport, wrap agar plates in parafilm and provide 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 | Phone (1-877-GENEWIZ (436-3949), Ext. 3350)