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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 s 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.

Technics

High throughput PCR

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

  • The presence or absence of a mutated allele
  • The approximate quantity (rare / abundant) of mutated allele

High throughput qPCR

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.

ddPCR

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.

  • Requires smaller sample size than other PCR technologies.
  • High throughput PCR.
  • Very precise quantification: sample division and counting of positive droplets over total droplets (+/- 5% over +/- 30% for qPCR) allow detection of low level differences.

PHENOMIN expertise