Authorized Reprint from Journal of Forensic Sciences. November 1997 ©Copyright 1997 American Society f or Testing and Materials. 100 Barr Harbor Drive, West Conshohocken. PA 19428-2959 Martin P. Evison, I Ph.D.; David M. Smillie, 2 Ph.D.; and Andrew T Chamberlain, 3 Ph.D. Extraction of Single-Copy Nuclear DNA from Forensic Specimens with a Variety of Postmortem Histories* REFERENCE: Evi son MP, Smilli e DM, Chamberlain AT. Extractio n of single-copy nucl ear DNA from specim n5 with a vari ety of postmorte m histories. J Forensic Sci 1997 ;42(6):1032- 1038. ABSTRACT: Specimens of human bo ne. tceth and dri ed hlood spot s fro l11 3 mo nth s to 91 years old, wi th a vari ety of postmortem histories. were used in a comparative study of recov ery of ingle- copy nucl ear DNA sequences fr om forens ic materi al. Sequences of the amelogenin and HLA· DPB I genes were chosen fo r their val ue in sex in g and identificatio n. Sequences of th e mitochondrial non·coding region V were also amp li fi ed to compare the recovery of mitochon dri al and single·copy nuclear DNA. A variation of th e silica method for DN A extraction was refined fo r appl ication to the fo re nsic spec ime ns in this samp le. Single·copy nucl ear DN A was amplified from 100% of re cent postopera tive bone specimens (n = 6) , 80% of for ens ic teeth an d bone pecimens (n = 10), 78 % of recentl y extracted teeth (n = 18 ), 78% of ex humed bone up to 91 ye ars old (n = 37) and 69% of 15 year ol d bone specimens fixed in 10% fo mlalin (n = 20). Amelog en in se xi ng was correc in 85% of cases (n = 74) in which th e sex of the donor had been recorded. There was no correlati on between th e age of the specimen and the extent of DN A prese rv ati on. KEYWORDS: forensic science, HLA· DPB I, amel ogeni n, DNA ext raction, PCR ; rn IDN A, bo ne; leeth, dri ed blood The sensitivity of the PCR tec hnj que is well kn own and its effectiveness in forensic analysis is well esta blished (1). Ap pli ca- tions to a variety of forensically- interesting material , such as hair (2), fi ngernails (3), dried blood (4), wax-emb edded tis sues (5,6), excrement (7), bone (8- 11 ) and teeth (12,1 3) have been demon- trated. Forensic DN A analysis may target nucl e ar loci (8- 11 , 14,1 5) or mitochon drial DNA (10,12), which occurs in mult iple copi es in the cell. Sequences f rom functi onal nu clear genes are a difficult target due to their low co py·number, but remain imp ortant in forensic analysis due to the high degree of polymorphism occurring in parts of the genome , no tably in the HL A c omplex (13,16,17), and because of the im portance of loci on the sex chro- mosomes in DNA sexing techniques (18). In this study we ha ve sought to c ompare the effectiveness of one method for single-copy nuclear DNA recove ry and analysis on 'Department of Forensic Pathology. Universi ty of Sheffield, Sh effield S3 7ES. UK. 2Trenl Centre, National Blood Service, Longley Lane, Sheffield. S5 71N, UK. .lDeparlmenl of Archaeology & PrehislOry, University of Sheffield, Shef· fi eld SI 4DT. UK. *This research was supponed by the Natural Environment Research Council. the UK National Blood Authority. and the University of Shef· field, UK. Received 26 Sept. 1996; and in revised form 5 Feb. 199 7; accepled 21 March 1997 . a varielY of for ensi c specimen types, focusing on the polymorphic HLA -DPB I locus and on the X-Y homol ogous amelogenin gene. We have also amplified the mi.lochondrial non-c ding region V [0 provide a co mpariso n of mi tochondrial and nucl ear DNA survival. The seque nces we target are all of a size which would reasonably be expected to survive in forensic spe cimens. Human skeletal material co mp rj sed bone recovered post- opera tively (n = 6); teeth extracted during dental treatment or shed deciduou teeth (n = 18); bo ne fix ed in fo on ahn (n = 20); and teeth and bone rec overed from forensic cases (n = 10) or as a result of exhumatio n (n = 37). There was a vari able degree of d ec omposit.ion and ske le tonization represe nted in t he sample, and some specimens had been subjected to preparat ive che mic al procedu res or buried in qu ick-lime. Dried blood spots (n = 28) were collected on gauze. Because of its value in remov ing peR inhib itors, we used a variation of the silica metho d of DNA extraction (1 9,20), which we ha ve ada pt ed to a r ange of forens ic material. Negative controls were included thro ughout in o rder to allow an assess ment of the exte nt and influ ence of contamin ation with extr aneous DNA . Materials and Methods Equipment Prepa ra tion Prec autions were taken ag ail1St contamination with intrusive DN A (21). Bone extraction equipment was cl eaned by soaking in 5.0% sodium hypoc hlorite solution for I h. Pre· and post-PCR activit ies were conducted in eparate roo ms and a laminar flow ca binet was used during xtraction and purificalion steps. Surfaces of bone or teeth were cleaned by washing with 0.5 % sodium hypo chlorite solution or by abrasion with a grinding tip or drill bit, which was then discarded. Sample Preparation Post · ope ratjve fra gments of femur and tibia were recovered followi ng routine orth opaedic surgery. Specimens of shaved bo ne were pul veri zed prior to extraction, except for one specimen, which was ex tracted fTom a femoral head by drilling with a flamed bit. Exhumed material came from three cemeteries. Burials had taken pla ce between 1904 and 1 984. Specimen Ai originated from a grave treated with quick-l ime (CaO) and subsequentl y contami· nated with automotive lubricating oil. Powdered bone from 16 of the e xhumed skeletons was generated during the removal of segments from the shafts of long - bone s by hacksaw for nitrogen analysis (see below). Cross -sect.ions of tibia had been removed from donated medical cadavers during 1980 and fixed in 10% fonn alin. Bone powder was extracted f rom the cross-sections by drilling with a flamed bit, material from the outer surface being 1032 Evison, M.P., Smillie, D.M. and Chamberlain, A.T. (1997). Extraction of single-copy genomic DNA from forensic specimens with a variety of post-mortem histories. Journal of Forensic Sciences, 42(6), 1032-8. ISSN 0022-1198 (Print).