Rolling Circle Amplification
DNA amplification by Rolling Circle Amplification (RCA PCR) is a method used to amplify whole circular genomes such as circular viral genomes, mitochondrial DNA, and microbial genomes up to 6.5 Mb. This method is based on the properties of Φ29 DNA polymerase. This enzyme has an important proofreading activity and a processivity allowing it to polymerize more than 70 000 nucleotides without detaching itself from the matrix DNA.
Thanks to this technique, SMALTIS proposes to specifically amplify by RCA your low amont of circular DNA markers present in your complex samples. The amplicons will then be detected and quantified specifically either by real-time PCR or by Southern blotting experiments.
According to your needs our staff will perform your project according to several steps:
STEP 1: Feasability Study
This step included a litterature review to analyse already published data and will allows our staff to:
- Design specific primers and probes
- Develop and set-up RCA method with your samples
- Determine the optimal amplification conditions
- Verify the specificity of primers and probes
- Associate the optimal detection method (qPCR or Southern Blot)
During this step, the analysis of a limited number of samples will also be done to validate the method. A detailed and illustrated report will be written to summarise the data.
STEP 2: Routine Analysis
This step allow us to analyse all your samples according to the previously validated method.
Examples of achivements:
- Detection of Human Endogenous virus HERV in cell pellets
- Detection and quantification of Human Endogenous virus HERV in blood samples collected in PAXGene Tubes.
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PCR & Sequencing
Quantification of gene expression