17 Such degradations and contaminations increase with the corpse decay and click here with post-mortem time span. Four different protocols were tested to recover DNA from pre-molar and molar teeth from 26 cadavers in bad decomposition stages with different post-mortem intervals. We compared the amount of DNA obtained and the DNA profiles with the time elapsed between

death and laboratory procedures. Forensic Laboratory of the Department of Legal Medicine of Instituto-Geral de Perícias (Rio Grande do Sul, Brazil) received 26 questioned samples that were unidentified due their advanced stage of decomposition. A task-force with the objective to resolve these 26 pending caseworks was carried out. Molar or premolar teeth were removed from corpses, cleaned with sterilized water only (we did not use abrasive, bleach, sandpaper, nor any mechanisms of deep cleaning), and stored for at least 24 h at −80 °C (SANYO UltraFreezer, Tokio, Japan). For each evaluated corpse data were recorded regarding subject’s age and sex, corpse condition, local where the corpses were found, and estimated post-mortem

time. The APO866 molecular weight four different protocols used to extract DNA are demonstrated in Fig. 1. Each tooth was grinded using IKA Works A11 Basic Analytical Mill (IKA® Processing Equipment), and the resulting powder was weighed in a precision balance (Adventure™; AR3130, OHAUS® Corp. pine Brook, NJ, USA) and separated into four 2 ml tubes. Around 0.6 g (max = 1.02 g; min = 0.34 g) of teeth powder was used for each DNA extraction. DNA was extracted using 600 μl of lysis buffer [100 mM NaCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 2% SDS (sodium dodecyl sulphate), 10 mM Tris–HCl (pH 8), 24 μl of 20 mg/ml proteinase K (Invitrogen, Carlsbad, USA), and 48 μl of 1 M DTT (dithiotreitol; Invitrogen, Carlsbad, CA, USA)]. Samples were Loperamide incubated at 56 °C for 2 or 12 h. For precipitation, 700 μl of UltraPure™ (phenol:chloroform:isoamyl alcohol, 25:24:1, Invitrogen, Carlsbad, CA, USA] were added, vortexed, and centrifuged for 7 min at 15,000 × g. The upper aqueous layer was placed inside

a Microcon™-100 concentrator (Millipore, Beverly, MA, USA) and centrifuged at 500 × g until only a few micro-liters remained. Microcon™-100 filtering was repeated twice by adding 400 μl of DNA-free H2O. Fifty micro liters of DNA-free H2O was added, the columns were inverted, and the kept was collected by centrifugation at 1000 × g for 3 min. The final sample was transferred into a new micro-centrifuge tube and stored at −20 °C. Alternatively, the upper aqueous layer was precipitated by adding an equal volume of isopropanol, centrifuged at 7000 × g for 7 min, and washed twice with 70% ethanol, centrifuged, and dried in a dry bath at 95 °C for 5 min followed by proteinase K inactivation at 95 °C for 5 min.