title: The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics.
abstract: The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 μg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.
authors.affiliation: Department of Medicine, Surgery and Health, University of Trieste, Trieste, Italy*
authors.forename: Paolo
authors.initials: P
authors.lastname: Fattorini
authors.forename: Carlo
authors.initials: C
authors.lastname: Previderè
authors.forename: Solange
authors.initials: S
authors.lastname: Sorçaburu-Cigliero
authors.forename: Giorgio
authors.initials: G
authors.lastname: Marrubini
authors.forename: Milena
authors.initials: M
authors.lastname: Alù
authors.forename: Anna M
authors.initials: AM
authors.lastname: Barbaro
authors.forename: Eugenia
authors.initials: E
authors.lastname: Carnevali
authors.forename: Angel
authors.initials: A
authors.lastname: Carracedo
authors.forename: Lucia
authors.initials: L
authors.lastname: Casarino
authors.forename: Lara
authors.initials: L
authors.lastname: Consoloni
authors.forename: Silvia
authors.initials: S
authors.lastname: Corato
authors.forename: Ranieri
authors.initials: R
authors.lastname: Domenici
authors.forename: Matteo
authors.initials: M
authors.lastname: Fabbri
authors.forename: Emiliano
authors.initials: E
authors.lastname: Giardina
authors.forename: Pierangela
authors.initials: P
authors.lastname: Grignani
authors.forename: Stefania Lonero
authors.initials: SL
authors.lastname: Baldassarra
authors.forename: Marco
authors.initials: M
authors.lastname: Moratti
authors.forename: Vanessa
authors.initials: V
authors.lastname: Nicolin
authors.forename: Susi
authors.initials: S
authors.lastname: Pelotti
authors.forename: Andrea
authors.initials: A
authors.lastname: Piccinini
authors.forename: Paola
authors.initials: P
authors.lastname: Pitacco
authors.forename: Laura
authors.initials: L
authors.lastname: Plizza
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Resta
authors.forename: Ugo
authors.initials: U
authors.lastname: Ricci
authors.forename: Carlo
authors.initials: C
authors.lastname: Robino
authors.forename: Luca
authors.initials: L
authors.lastname: Salvaderi
authors.forename: Francesca
authors.initials: F
authors.lastname: Scarnicci
authors.forename: Peter M
authors.initials: PM
authors.lastname: Schneider
authors.forename: Gregorio
authors.initials: G
authors.lastname: Seidita
authors.forename: Lucia
authors.initials: L
authors.lastname: Trizzino
authors.forename: Chiara
authors.initials: C
authors.lastname: Turchi
authors.forename: Stefania
authors.initials: S
authors.lastname: Turrina
authors.forename: Paolo
authors.initials: P
authors.lastname: Vatta
authors.forename: Carla
authors.initials: C
authors.lastname: Vecchiotti
authors.forename: Andrea
authors.initials: A
authors.lastname: Verzeletti
authors.forename: Francesco
authors.initials: F
authors.lastname: De Stefano
mesh_terms: D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D016172:DNA Fingerprinting / Q000379:methods; D053803:Forensic Genetics / Q000379:methods* / Q000592:standards*; D060005:Genotyping Techniques; D006801:Humans; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results
chemical_list: D004247:DNA
keywords: DNA depurination; Forensic genetics; PCR fidelity; STR typing