Amplification of nucleic acids

The amplification of nucleic acids by PCR (Polymerase Chain Reaction), is the reference method to obtained large quantities of a specific DNA sequence from a sample of nucleic acids.
Thanks to this method and our standard operation procedures, SMALTIS proposes to amplify your sequences of interest and to sequence them, to quantify the expression of your target genes or to specifically amplify your circular DNA molecules by RCA PCR.

PCR & Sequencing

Amplification sequencing of DNA fragments is a reference method for many applications such as the study of the genetic polymorphism of a DNA target, identification of mutations conferring particular phenotypes or verification of plasmid DNA sequences. 
Regardless the nature of your samples (plasmids, blood, cell pellets, microorganisms, ...) SMALTIS offers you to control your sequences of interest, from the purification of nucleic acids to the alignment of nucleotide sequences via the design and the synthesis of specific primers. The services also include various complementary services such as agarose gel purification or chromosome-walking.

At the end of the service, a detailed and illustrated report will be sent to you.

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Quantification of gene expression

Gene expression analysis is fundamental in many fields of research and allows to understand dynamic changes in a bacterium, cell, tissue or organism. Indeed, if the genome is identicial in each cells of a an organism, genes can be expressed in a specific and different manners, depending on time (specific to development stage), on space (expression specific to a cell type or tissue) and/or on characteristics of a given state (normal, pathological, in response to stimuli, ...).
SMALTIS proposes to specifically study the expression of your genes by quantifying the number or your targetted mRNA thanks to its real-time PCR platform. 

Depending on your needs, our staff will carry out your project following several steps:

Step 1: Feasability Study

This step includes a litterature review to analyse already publised data allowing our staff to: 
      - Identify existing commercial quantification kits
      - Design specific primers and probes (SybrGreen or TaqMan technologies)
      - Develop a qPCR protocol
      - Determine the optimal amplification conditions
      - Verify the specificity of primers and probes
      - Validate the choice of housekeeping genes

During this step, the analysis of a limited number os 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
Our platform equipped with a RotorGene 6000 and CFX Connect allows us to process a few samples at several hundred simultaneously.

Examples of achievement

- Expression analysis of virulence and resistance genes in strains of Klebsiella pneumoniae
- Study of the inductive effect of antibiotics on the expression of efflux systems in Pseudomonas aeruginosa
- Detection of episomal DNA in blood samples collected using PAXGene® Technology
- Gene expression analysis of genes encoding IL-6, IL-8 and TNFα cytokines in cells previously infected with different bacterial strains
- Quantification of several genes in transfected cell lines.

More information 


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 allows us to analyse all your samples according to the previously validated method.

Examples of achievement

- Detection of Human Endogenous virus HERV in cell pellets
- Detection and quantification of Human Endogenous virus HERV in blood samples collected in PAXGene Tubes®