Technical Note Kerry L. Opel, 1 M.A.; Denise T. Chung, 2,5 Ph.D.; Jirˇı´ Dra´bek, 2,6 Ph.D.; Nancy E. Tatarek, 3 Ph.D.; Lee Meadows Jantz, 4 Ph.D.; and Bruce R. McCord, 1 Ph.D. The Application of Miniplex Primer Sets in the Analysis of Degraded DNA from Human Skeletal Remains à ABSTRACT: A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50–280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex s 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Pow- erplex s 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains. KEYWORDS: forensic science, multiplex PCR, short tandem repeat, STR, nuclear DNA, DNA typing, human skeletal remains DNA template used for short tandem repeat (STR) profiling in forensic casework may become highly degraded because of bac- terial, biochemical, or oxidative processes (1). In these circum- stances, the possibility of finding an intact target sequence is greatly reduced because of extensive template fragmentation. Be- cause of this problem, it is often not practical to use STRs to analyze such samples. Instead, mitochondrial (mt) DNA can be used. As mtDNA is present in large numbers in cells, extraction and analysis of this type of DNA has a higher probability of pro- ducing typable results (2). However, mtDNA has the disadvan- tages of haploid inheritance, low discriminatory power, and increased analysis time. Genotyping with STR loci produces results quickly, and with high discriminatory power, yet there is a need to extend this tech- nique to access degraded samples. Commercial multiplex STR kits typically have amplicon sizes ranging from 100 to 480 base pairs (bp) (3–5). Because of this wide range of amplicon sizes, allele dropout for the larger sized amplicons often results in a partial genetic profile when these kits are used to amplify highly degraded DNA samples (3,5). There are also problems because of contamination from bacteria and polymerase inhibitors from soil (6–8). Sample concentration has also been a issue, as 1–5 g of bone powder may be required to obtain sufficient DNA for anal- ysis, and removal of this much material from the bone can be very destructive to samples that must be kept for forensic or archival purposes (6,9–11). To help alleviate the problems associated with analyzing DNA from degraded samples, a new set of STR primers known as Miniplexes have been recently designed. The primers were cre- ated by moving the primer binding sites as close as possible to the repeat region (12,13). Previous examples of the application of re- duced amplicon size for various STR loci have already been pub- lished (14–16), but these attempts have generally focused on single loci and not the full set of 13 CODIS loci recommended by the United States Department of Justice for profile data-basing (12). The Miniplexes cover 12 of the 13 CODIS loci, with three loci per set. To avoid overlapping loci, only one locus could be assigned to each dye lane for detection. However, the size ranges for two of these sets, Miniplex 1 and Miniplex 3, did not overlap, so the two sets were combined to form a six loci (in 4 dye de- tection) multiplex set known as ‘‘Big Mini.’’ In a previous study (13), it was demonstrated that the efficiency of amplification of larger sized template DNA is greatly reduced in artificially (enzymatically) degraded samples, and that the Miniplexes were able to amplify the degraded DNA to produce 5 Present Address: Center for Neurological Diseases, Brigham & Wom- en’s Hospital, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur Room, Boston, MA 02115-5817. 6 Present Address: Palacky University, Department of Biochemistry, Olomouc, CZ-783 71, Czech Republic. à This project was supported under award 2002-IJ-CX-K007 from the National Institute of Justice and by the Provost Undergraduate Research Fund of Ohio University. 1 Department of Chemistry and Biochemistry, Florida International Uni- versity, University Park, 11200 SW, FL 33199. 2 Department of Chemistry and Biochemistry, Ohio University, 136 Clippinger Laboratories, Athens, OH 45701. 3 Department of Sociology and Anthropology, Ohio University, 111 Bentley Annex, Athens, OH 45701. 4 Department of Anthropology, University of Tennessee, Knoxville, TN 37996. Received 3 April 2005; and in revised form 25 June and 9 Sept. 2005; ac- cepted 1 Oct. 2005; published 14 Feb. 2006. 351 Copyright r 2006 by American Academy of Forensic Sciences J Forensic Sci, March 2006, Vol. 51, No. 2 doi:10.1111/j.1556-4029.2006.00077.x Available online at: www.blackwell-synergy.com