title: A simple method for extracting DNA from old skeletal material.
abstract: Extraction of DNA from old skeletal material is of great importance in the identification of human remains, but is particularly difficult because the methods currently employed, especially those using phenol/chloroform, are not always satisfactory. A simple technique based on the removal of non-nucleic acid material by salting out (precipitation) with saturated sodium acetate is described; the presence of DNA in the extract being confirmed by amplification of selected sequences of the HLA-DRB1 gene using the polymerase chain reaction (PCR). The method was applied to fresh bone (five femoral heads and six vertebral bodies) and to bone from two forensic cases, 3 and 9 months post-mortem, respectively. Parallel extractions using the phenol/chloroform technique were performed on all samples in order to compare the efficiency of the two methods. Using sodium acetate precipitation, amplifiable DNA was consistently extracted from fresh bone, as well as from the two forensic cases. With the phenol/chloroform method, amplification was successful in only 7 out of 11 instances with the fresh bone samples and failed in both forensic cases. The studies also showed that an effective way of removing PCR inhibitors is to subject the extract to agarose gel electrophoresis, isolate the high molecular weight area and re-extract the DNA from the gel by boiling. It was concluded that the sodium acetate method is a valid alternative to established techniques for extracting DNA from bone and that it offers the advantages of being simple, quick, inexpensive and avoids using hazardous reagents.
authors.affiliation: Trent Blood Transfusion Centre, Sheffield, UK.
authors.forename: C
authors.initials: C
authors.lastname: Cattaneo
authors.forename: D M
authors.initials: DM
authors.lastname: Smillie
authors.forename: K
authors.initials: K
authors.lastname: Gelsthorpe
authors.forename: A
authors.initials: A
authors.lastname: Piccinini
authors.forename: A R
authors.initials: AR
authors.lastname: Gelsthorpe
authors.forename: R J
authors.initials: RJ
authors.lastname: Sokol
mesh_terms: D000085:Acetates; D019342:Acetic Acid; D000328:Adult; D001483:Base Sequence; D001842:Bone and Bones / Q000737:chemistry*; D002725:Chloroform; D004247:DNA / Q000302:isolation & purification*; D018732:Forensic Anthropology / Q000379:methods*; D006684:HLA-DR Antigens / Q000235:genetics*; D059811:HLA-DRB1 Chains; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012997:Solvents
chemical_list: D000085:Acetates; D006684:HLA-DR Antigens; D059811:HLA-DRB1 Chains; D012997:Solvents; D002725:Chloroform; D004247:DNA; D019342:Acetic Acid