44(3):273-280,2003 FORENSIC SCIENCES Specific Quantification of Human Genomes from Low Copy Number DNA Samples in Forensic and Ancient DNA Studies Antonio Alonso, Pablo Martín, Cristina Albarrán, Pilar García, Dragan Primorac 1 , Oscar García 2 , Lourdes Fernández de Simón, Julia García-Hirschfeld, Manuel Sancho, Jose Fernández-Piqueras 3 Instituto Nacional de Toxicología, Servicio de Biología, Madrid, Spain; 1 Laboratory for Clinical and Forensic Genetics, Split University Hospital, Split, Croatia; 2 Area de Laboratorio Ertzaintza, Bilbao; and 3 Universidad Autonoma de Madrid, Laboratorio de Genética Molecular Humana, Cantoblanco, Madrid, Spain We reviewed the current methodologies used for human DNA quantitation in forensic and ancient DNA studies, in- cluding sensitive hybridization methods based on the detection of nuclear alpha-satellite repetitive DNA regions or more recently developed fluorogenic real-time polymerase chain reaction (PCR) designs for the detection of both nu- clear and mitochondrial DNA regions. Special emphasis has been put on the applicability of recently described differ- ent real-time PCR designs targeting different fragments of the HV1 mtDNA control region, and a segment of the X-Y ho- mologous amelogenin gene. The importance of these quantitative assays is to ensure the consistency of low copy number DNA typing (STR profiling and mtDNA sequencing). Key words: DNA; DNA fingerprinting; DNA mitochondrial; evolution; forensic medicine; polymerase chain reaction; tan- dem repeat sequences The specific quantification of human DNA mole- cules has gained great importance in forensic (1-3) and ancient DNA studies (4,5) as aid in the interpreta- tion of the consistency and the reliability of polymer- ase chain reaction (PCR)-based short tandem repeat (STR) profiling and mitochondrial DNA (mtDNA) se- quence analysis from low copy number DNA sam- ples (6-10). First, an estimation of the quantity of hu- man genome is a recommended procedure in foren- sic casework (11,12) that will aid to decide whether the isolated DNA is suitable for nuclear or mitochon- drial DNA analysis and to adjust the DNA input to im- prove the performance of subsequent end-point PCR- based DNA typing approaches. Second, classification of low copy number DNA samples by DNA content allowed decision to be made on the number of DNA typing repetitions required for statistically reliable re- sults (13). In particular, it is important to consider three types of errors that could affect DNA typing reli- ability when low copy number DNA samples are ana- lyzed: allele drop-out, stutter false alleles, and the in- fluence of low levels of DNA contamination (8-10). Third, DNA quantification ensures the optimal use of the limited amounts of DNA found in many forensic evidences ensuring that the DNA is not wasted with expensive repetitive endpoint PCR typing analysis performed with inappropriate amounts of DNA tem- plate. Furthermore, some DNA quantification designs could also provide additional information about DNA degradation (14,15), the presence of PCR inhibitors (3), or specific information about the DNA content of X and Y sexual chromosomes (14-17). We reviewed the most recent methodologies used for human DNA quantitation in forensic and an- cient DNA studies, including sensitive hybridization methods based on the detection of nuclear alpha-sat- ellite repetitive DNA regions (1,18) or more recently developed fluorogenic real-time PCR designs for the detection of both single-copy nuclear genes and mtDNA regions (3,14-17). Special emphasis has been put on the applicability of different real-time PCR de- signs recently described by the authors targeting dif- ferent fragments of the hypervariable (HV)1 mtDNA control region, and a segment of the X-Y homologous Amelogenin (AMG) gene (14,15). Nuclear DNA Quantification Detection of Highly Repetitive Human DNA Sequences by Molecular Hybridization At present, the most popular method for quantifi- cation of picogram quantities of the nuclear human DNA in forensic genetics is the slot-blot hybridization approach (1,18) This method allows detection of highly repetitive satellite DNA regions with the pri- mate-specific alpha-satellite probe to the D17Z1 lo- cus (19). Early development of a commercial chemil- uminescent human DNA quantification kit (20) to tar- www.cmj.hr 273