3 functional genes categories have been mapped : Dog Genes, TOASTs and ESTs.
- Dog gene sequences analysis enable us to design primers which characterize the gene locus.
- Analysis of several mammalian gene sequences allows to derive orthologous primers usable for dog.
These markers are called TOAST for Traced Orthologous Amplified Sequence Tag.
- Human ESTs from Généthon amplified on dog DNA but not on hamster DNA have also been mapped.
Type I markers typing:
- Every markers are typed on a panel consisting of 126 independant PCR amplifications plus controls.
- PCR products are visualized onto stained agarose gel and captured by a CCD picture system.
- Data are then captured by a semi-automated fashion through a program developed in the lab and transfered to a UNIX workstation.
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- (CA) type microsatellites were identified and analyzed for size polymorphism in the lab.
- They were screened with a biotinylate CA probe followed by a chemiluminescent detection method.
- Polymorphism was analyzed on 377 ABI sequencers.
- Primers are then designed to characterize polymorphic microsatellite locus.
Type II markers typing :
- A fluorescent method has been developed in the lab consisting of CA fluorescent probe incorporation that is quantified during PCR amplification.
- Data are then collected directly by integrating and calculating the PCR tube light emission.
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