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Comparison of extraction methods for the recovery, amplification and species‐specific analysis of DNA from bone and bone meals
Author(s) -
Prado Marta,
Franco Carlos M.,
Fente Cristina A.,
Cepeda Alberto,
Vázquez Beatriz I.,
BarrosVelázquez Jorge
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200204)23:7/8<1005::aid-elps1005>3.0.co;2-1
Subject(s) - ethylenediaminetetraacetic acid , dna , chromatography , polymerase chain reaction , chemistry , dna extraction , extraction (chemistry) , restriction fragment length polymorphism , microbiology and biotechnology , sodium dodecyl sulfate , restriction enzyme , proteinase k , biology , biochemistry , gene , chelation , organic chemistry
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°C or 100°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 νg, and lasting from 1 to 3 h) at 65°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°C for 1 h, this being followed by treatment with 800 νg 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 Pal I and Mbo I.