title: Comparison of extraction methods for the recovery, amplification and species-specific analysis of DNA from bone and bone meals.
abstract: 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.
authors: Area de Nutrición y Bromatología, Departmento de Química Analitica, Nutrición y Bromotología, Facultada de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain.
authors: Marta
authors: M
authors: Prado
authors: Carlos M
authors: CM
authors: Franco
authors: Cristina A
authors: CA
authors: Fente
authors: Alberto
authors: A
authors: Cepeda
authors: Beatriz I
authors: BI
authors: Vázquez
authors: Jorge
authors: J
authors: Barros-Velázquez
mesh_terms: 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
chemical_list: D001688:Biological Products; D008903:Minerals; D004247:DNA; C027762:bone meal