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Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence. This step is crucial in the model creation process as it allows to select animals carrying the good combination of alleles for phenotyping analyses. The different technics are based on DNA quantification for a specific sequence with a predefined length.
We offer customized genotyping services for knock-out, knock-in, transgenic or any other genetically modified murine model. Different sample types can be processed, including tails biopsies, ear tags and embryonic yolk sac.
All genotyping steps are covered: a complete design, optimization, verification of PCR amplification conditions for new or pre-existing strategies, genomic DNA extraction, PCR and/or qPCR reactions, analysis and sequencing for the gene of interest.
The fully automated service allows a high-throughput process. All used technologies are driven by in-house databases insuring integrated data management. We provide a detailed genotyping protocol to all our users on request.
Before you even begin any experiment, genotyping is the first thing you have to do with any genetically modified animal. This analysis is generally wrongly considered a basic task but in fact genotyping can be much more complex than expected. Poor genotyping can influence the biological conclusions of a study and basic research reproducibility1. Genotyping errors are one of the factors that explain that 15% of lines deposited to public repositories do not carry the mutation specified by the depositor2. Genotyping failures lead to a waste of time and money for research laboratories. In their recent paper3, our collaborators from PHENOMIN-ICS went back to PCR genotyping basic to provide Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High-Throughput Genotyping Protocol for Any Type of Mutation.
1.Pompanon, F., Bonin, A., Bellemain, E. & Taberlet, P. Genotyping errors: causes, consequences and solutions. Nat. Rev. Genet. 6, 847–859 (2005). 2.Lloyd, K., Franklin, C., Lutz, C. & Magnuson, T. Reproducibility: Use mouse biobanks or lose them. Nature 522, 151–153 (2015). 3.Jacquot, S., Chartoire, N., Piguet, F., Hérault, Y. & Pavlovic, G. Optimizing PCR for Mouse Genotyping: Recommendations for Reliable, Rapid, Cost Effective, Robust and Adaptable to High‐Throughput Genotyping Protocol for Any Type of Mutation. Curr. Protoc. Mouse Biol. 9, (2019).
The polymerase chain reaction (PCR) overexpresses the target DNA sequence over about a billion copies to obtain enough material for further use. Resulting products are then migrated on agarose gel which enables i) to infer the absence / presence of target DNA (if appropriate controls have been realized) ii) to vizualise the size of amplified DNA. These two elements are sometimes accompanied by Sanger sequencing; all together, this allows the detection of
Quantitative PCR (qPCR, or real time PCR) allows the determination of initial amount of a particular DNA sequence. The quantity of DNA is followed all along the reaction thanks to a fluorescent marker integrated into PCR products; the complete kinetics obtained at the end of the reaction allows the precise quantification of initial DNA sequence.
Traditional qPCR workflows include a multitude of steps that limit throughput, efficiency and scalability. To speed up data generation and reduce the pieptting- and operator- dependent variability, PHENOMIN is equiped with a fully automated platform for qPCR.
Droplet Digital PCR (ddPCR) is based on sample division into thousands of nanoliter-sized droplets using a water-oil emulsion droplet system; PCR amplification (and integration of fluorescent products) occurs in each individual droplet, i.e. thousands of independent amplification events are realized from a single sample. Each droplet is analyzed over fluorescence to determine the fraction of PCR-positive droplets in the original sample.