Comparison of extraction methods for the recovery, amplification and species-specific analysis of DNA from bone and bone meals.
We report the effect of several parameters on the efficiency of recovery of DNA from animal bones. The effects of preheating the samples (at either 60 degrees C or 100 degrees C) at different intervals (from 1 h to overnight) in different media (water, 0.5 M ethylenediaminetetraacetic acid (EDTA), or 0.5 M EDTA + 0.05% sodium dodecyl sulfate (SDS) were investigated. The effect of slight (5 min) or intense (30 min) pretreatments with ultrasound was also evaluated. Several different treatments with proteinase K (ranging from 200 to 800 microg, and lasting from 1 to 3 h) at 65 degrees C were also considered. Additionally, two different DNA extraction methods (based on silica resins and purification columns, respectively) were evaluated. The recovery of DNA from the samples was 40% higher when the bones were preheated in 0.5 M EDTA at 60 degrees C for 1 h, this being followed by treatment with 800 microg of proteinase K for 3 h. The DNA thus obtained was successfully amplified by polymerase chain reaction (PCR) using a set of primers specific to a 359 bp region of the mitochondrial cytochrome b gene, and the species of origin were identified by visualizing the restriction fragment length polymorphism (RFLP) with the endonucleases PalI and MboI.
Prado
Marta
M
Area de Nutrición y Bromatología, Departmento de Química Analitica, Nutrición y Bromotología, Facultada de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain.
Franco
Carlos M
CM
Fente
Cristina A
CA
Cepeda
Alberto
A
Vázquez
Beatriz I
BI
Barros-Velázquez
Jorge
J
D000818:Animals; D001688:Biological Products; D001842:Bone and Bones / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D008903:Minerals / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013045:Species Specificity
D001688:Biological Products; D008903:Minerals; D004247:DNA; C027762:bone meal