The amplification of nucleic acids by polymerase chain reaction, more commonly called PCR (Polymerase Chain Reaction) is the reference molecular biology technique allowing to obtain, from a nucleic acid sample, a large number of identical copies of a DNA fragment. Thanks to this technique, SMALTIS proposes to amplify and thus to detect the presence of DNA fragments of interest or of circular DNA molecules.
The use of DNA intercalating agents or fluorescent probes during the PCR reaction also allows to quantify in real time the increase in the number of newly synthesized DNA copies. Through this technique, called qPCR (quantitative PCR), SMALTIS can detect and quantify specifically a target of interest or the expression of target genes.
Our offer
Amplification by PCR and sequencing
amplification and sequencing of DNA fragments has many applications such as the study of the genetic polymorphism of a target of interest, the identification of mutations conferring particular phenotypes or the validation of plasmid DNA sequences.
Whatever your samples (plasmids, blood, cell pellets, microorganisms…), Smaltis offers you its services to control your sequences of interest, from nucleic acid purification to nucleotide sequence alignment, including the design and synthesis of specific primers.
Services also include various complementary services such as agarose gel purification or chromosome walking.
Quantification of gene expression
Gene expression analysis is fundamental in many fields of research and allows to understand dynamic changes in a bacterium, eukaryotic 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 manner, 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 offers to study specifically the expression of genes of interest by quantifying the number of messengers (mRNA) thanks to its Real-Time PCR platform, after a Reverse Transcription step allowing the conversion of mRNA into complementary DNA (cDNA).
According to your needs, our team will carry out your project following several steps:
Step 1: Feasability Study
This step includes a literature review to analyze existing data and a bioinformatics analysis of sequences of interest.
This will allow our experts to:
– Identify existing commercial quantification kits or design specific primers and probes (SybrGreen® or TaqMan® technologies)
– Determine the housekeeping genes
– Develop the 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 of samples will also be done to validate the method.
Step 2: Routine Analysis
This step allows us to analyse all your samples according to the previously validated method.
Our platform equipped with a RotorGene 6000 and a CFX Connect allows us to process a few samples to 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 eukaryotic cell lines.
Amplification of circular DNA molecules by 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 team will perform your project according to several steps:
Step 1: Feasability Study
This step includes a literature review to analyze existing data and a bioinformatics analysis of sequences of interest.
This will allow our experts 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 and quantification method
During this step, the analysis of a limited number of samples will also be done to validate the method.
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®.
