Molecular beacon genotyping for globoid cell leukodystrophy from hair roots in the twitcher mouse and rhesus macaque.
Rapid and accurate genotype determination is ideal for the maintenance of breeding colonies of laboratory animal models of genetic disease. The rhesus macaque and murine (twitcher) models of globoid cell leukodystrophy have a dinucleotide deletion or single nucleotide substitution, respectively, which abolish ceramide beta-galactosidase activity and are authentic models of Krabbe disease. We report a molecular beacon PCR assay for each species which allows unambiguous determination of the genotype in under 4h. The assay works reliably with DNA extracted from hair roots using Chelex-100 in a 20 min, 100 degrees C incubation. We demonstrate that genotyping from hair roots is a preferred alternative to collecting blood or tissue for DNA extraction because it reduces animal distress, uses an inexpensive reagent, and is simpler and faster. Following amplification on a standard thermocycler with a 96-well plate format, these molecular beacon assays can be read on a standard laboratory fluorescent plate reader, eliminating the need to use a real-time thermocycler or to open the plate for subsequent restriction enzyme digestion and gel electrophoresis. The multiplexed ratio of fluorescence from wild-type- and mutant-specific beacons reporting at 560 nm and 535 nm wavelengths is distinct for each genotype.
Terrell
Kimberly A
KA
Department of Biology, University of New Orleans, 2000 Lakeshore Drive, New Orleans, LA 70148, USA. kterrell@uno.edu
Rasmussen
Terri A
TA
Trygg
Cyndi
C
Bunnell
Bruce A
BA
Buck
Wayne R
WR
D000818:Animals; D004195:Disease Models, Animal; D005698:Galactosylceramidase / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics; D005821:Genetic Techniques; D005838:Genotype; D006197:Hair / Q000201:enzymology*; D007965:Leukodystrophy, Globoid Cell / Q000235:genetics* / Q000473:pathology*; D008253:Macaca mulatta; D051379:Mice; D008818:Mice, Neurologic Mutants; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D016324:Sequence Tagged Sites
D005819:Genetic Markers; D005698:Galactosylceramidase