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Rolling Circle Amplification (RCA) : Concatamers

Rolling Circle Amplification Important technique of Protein engineering Basic understanding of protein engineering

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Adena Cleris
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17 views2 pages

Rolling Circle Amplification (RCA) : Concatamers

Rolling Circle Amplification Important technique of Protein engineering Basic understanding of protein engineering

Uploaded by

Adena Cleris
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Rolling Circle Amplification (RCA)

This method is based on rolling circle amplification (RCA), which relies on the natural mechanism of
rolling circle replication used by cells to amplify circular DNA like plasmids.

The amplification process results in linear DNA duplexes containing tandem repeats of circular DNA
called concatamers, which can be directly transformed into a variety of strains.

Steps Involved in the Method

Cloning of Template Sequence

 Clone the template sequence into an appropriate plasmid.

Amplification Process

 Amplify the cloned sequence using random hexamer primers and Ø29 DNA polymerase
under error-prone rolling circle amplification (ep-RCA) conditions.

Addition of MnCl2

 Add MnCl2 to the reaction mixture of RCA. MnCl2 induces random point mutations in DNA
strands.

Transformation

 Transform the amplified product (DNA duplexes with random mutations) directly into the
host cells.

Screening

 Screen the host cells for the desired clone.

Standard ep-RCA Conditions

 Template DNA concentration: 1.5 pM


 MnCl2 concentration: 1.5 mM
 Reaction time: 24 hours
 Mutation rate: One amino acid per kilobase

Increasing the Mutation Rate

 Increase the concentration of MnCl2.


 Decrease the concentration of the template DNA.

Advantages of ep-RCA Over Error-Prone PCR

 No Need for Specific Primers


o Universal random hexamer primers can be used with any template.
 Isothermal Reaction
o No need to manage thermal cycling conditions.
 No Need for Post-Amplification Treatments
o No requirement for ligases or restriction endonucleases to treat amplified DNA
products.
 Practical Application
Fujji et al. used this method to enhance the ceftazidime resistance of TEM-1 β-lactamase.

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