Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies Antonio Alonso a,* , Pablo Martı ´n a , Cristina Albarra ´n a , Pilar Garcı ´a a , Oscar Garcı ´a b , Lourdes Ferna ´ndez de Simo ´n a , Julia Garcı ´a-Hirschfeld a , Manuel Sancho a , Concepcio ´n de la Ru ´a c , Jose Ferna ´ndez-Piqueras d a Instituto Nacional de Toxicologı ´a, Servicio de Biologı ´a, Luis Cabrera 9, 28002 Madrid, Spain b Area de Laboratorio Ertzaintza, Bilbao, Spain c Dpto. Biologı ´a Animal y Gene ´tica, Facultad de Ciencias, Universidad del Paı ´s Vasco, Bilbao, Spain d Laboratorio de Gene ´tica Molecular Humana, Universidad Autonoma de Madrid, Cantoblanco, Madrid, Spain Received 15 September 2003; accepted 3 October 2003 Abstract We explore different designs to estimate both nuclear and mitochondrial human DNA (mtDNA) content based on the detection of the 5 0 nuclease activity of the Taq DNA polymerase using fluorogenic probes and a real-time quantitative PCR detection system. Human mtDNA quantification was accomplished by monitoring the real-time progress of the PCR-amplification of two different fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This mtDNA real-time PCR design has been used to assess the mtDNA preservation (copy number and degradation state) of DNA samples retrieved from 500 to 1500 years old human remains that showed low copy number and highly degraded mtDNA. The quantification of nuclear DNA was achieved by real-time PCR of a segment of the X–Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and a X-specific fragment (AMGX: 106 bp) making possible not only haploid or diploid DNA quantitation but also sex determination. The AMG real-time PCR design has been used to quantify a set of 57 DNA samples from 4–5 years old forensic bone remains with improved sensitivity compared with the slot–blot hybridization method. The potential utility of this technology to improve the quality of some PCR-based forensic and ancient DNA studies (microsatellite typing and mtDNA sequencing) is discussed. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Ancient DNA; Forensic genetics; Real-time PCR; Mitochondrial DNA control region; Amelogenin gene 1. Introduction Human DNA quantification has became an essential analysis to ensure the quality of PCR-based studies per- formed from low copy number and/or highly damaged DNA samples that are analyzed in a routine way by different scientific disciplines including ancient DNA studies [1,2], molecular ecology [3], or forensic genetics [4]. Random allele dropout [5] is a very common PCR artifact when microsatellite typing is performed from low copy number samples like non-invasive shed hairs and fecal material [3], bones [4], or other forensic samples [6]. This could lead to false homozygous typing results. In vitro nucleotide mis- sincorporation could also have a great impact in the accuracy of mitochondrial DNA (mtDNA) sequence analysis if PCR-amplifications start from very few copies or even single DNA molecules [1]. This scenario could be further compli- cated if samples are also subjected to degradation, to base pair modifications [7], or to different contamination levels. Therefore, the estimation of the number of human DNA template molecules is very useful and can be accomplished by high sensitive hybridization methods with primate Forensic Science International 139 (2004) 141–149 * Corresponding author. Tel.: þ34-9-1562-9190; fax: þ34-9-1563-6924. E-mail address: a.alonso@mju.es (A. Alonso). 0379-0738/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2003.10.008