The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

Gregorio Seidita, Andrea Verzeletti, Eugenia Carnevali, Francesca Scarnicci, Ranieri Domenici, Lara Consoloni, Anna M. Barbaro, Paola Pitacco, Solange Sorçaburu-Cigliero, Silvia Corato, Marco Moratti, Matteo Fabbri, Peter M. Schneider, Luca Salvaderi, Paolo Fattorini, Emiliano Giardina, Lucia Trizzino, Laura Plizza, Chiara Turchi, Vanessa NicolinPierangela Grignani, Paolo Vatta, Milena Alù, Stefania Lonero Baldassarra, Lucia Casarino, Carlo Previderè, Stefania Turrina, Susi Pelotti, Nicoletta Resta, Andrea Piccinini, Carla Vecchiotti, Ugo Ricci, Carlo Robino, Francesco De Stefano, Anna M. Barbaro, Angel Carracedo, Giorgio Marrubini

Risultato della ricerca: Article

7 Citazioni (Scopus)

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 (r2 = 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.
Lingua originaleEnglish
pagine (da-a)3134-3144
Numero di pagine11
RivistaElectrophoresis
Volume35
Stato di pubblicazionePublished - 2014

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Forensic Genetics
Reproducibility of Results
DNA
Amplification
Genotype
Alleles
Allelic Imbalance
Polymerase Chain Reaction
Multiplex Polymerase Chain Reaction
Documentation
Artifacts
DNA Damage
Genetics
Hydrolysis
Molecular Weight
Molecular weight
Tissue
Incidence

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

Cita questo

Seidita, G., Verzeletti, A., Carnevali, E., Scarnicci, F., Domenici, R., Consoloni, L., ... Marrubini, G. (2014). The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics. Electrophoresis, 35, 3134-3144.

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics. / Seidita, Gregorio; Verzeletti, Andrea; Carnevali, Eugenia; Scarnicci, Francesca; Domenici, Ranieri; Consoloni, Lara; Barbaro, Anna M.; Pitacco, Paola; Sorçaburu-Cigliero, Solange; Corato, Silvia; Moratti, Marco; Fabbri, Matteo; Schneider, Peter M.; Salvaderi, Luca; Fattorini, Paolo; Giardina, Emiliano; Trizzino, Lucia; Plizza, Laura; Turchi, Chiara; Nicolin, Vanessa; Grignani, Pierangela; Vatta, Paolo; Alù, Milena; Baldassarra, Stefania Lonero; Casarino, Lucia; Previderè, Carlo; Turrina, Stefania; Pelotti, Susi; Resta, Nicoletta; Piccinini, Andrea; Vecchiotti, Carla; Ricci, Ugo; Robino, Carlo; De Stefano, Francesco; Barbaro, Anna M.; Carracedo, Angel; Marrubini, Giorgio.

In: Electrophoresis, Vol. 35, 2014, pag. 3134-3144.

Risultato della ricerca: Article

Seidita, G, Verzeletti, A, Carnevali, E, Scarnicci, F, Domenici, R, Consoloni, L, Barbaro, AM, Pitacco, P, Sorçaburu-Cigliero, S, Corato, S, Moratti, M, Fabbri, M, Schneider, PM, Salvaderi, L, Fattorini, P, Giardina, E, Trizzino, L, Plizza, L, Turchi, C, Nicolin, V, Grignani, P, Vatta, P, Alù, M, Baldassarra, SL, Casarino, L, Previderè, C, Turrina, S, Pelotti, S, Resta, N, Piccinini, A, Vecchiotti, C, Ricci, U, Robino, C, De Stefano, F, Barbaro, AM, Carracedo, A & Marrubini, G 2014, 'The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics', Electrophoresis, vol. 35, pagg. 3134-3144.
Seidita, Gregorio ; Verzeletti, Andrea ; Carnevali, Eugenia ; Scarnicci, Francesca ; Domenici, Ranieri ; Consoloni, Lara ; Barbaro, Anna M. ; Pitacco, Paola ; Sorçaburu-Cigliero, Solange ; Corato, Silvia ; Moratti, Marco ; Fabbri, Matteo ; Schneider, Peter M. ; Salvaderi, Luca ; Fattorini, Paolo ; Giardina, Emiliano ; Trizzino, Lucia ; Plizza, Laura ; Turchi, Chiara ; Nicolin, Vanessa ; Grignani, Pierangela ; Vatta, Paolo ; Alù, Milena ; Baldassarra, Stefania Lonero ; Casarino, Lucia ; Previderè, Carlo ; Turrina, Stefania ; Pelotti, Susi ; Resta, Nicoletta ; Piccinini, Andrea ; Vecchiotti, Carla ; Ricci, Ugo ; Robino, Carlo ; De Stefano, Francesco ; Barbaro, Anna M. ; Carracedo, Angel ; Marrubini, Giorgio. / The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics. In: Electrophoresis. 2014 ; Vol. 35. pagg. 3134-3144.
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T1 - The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics

AU - Seidita, Gregorio

AU - Verzeletti, Andrea

AU - Carnevali, Eugenia

AU - Scarnicci, Francesca

AU - Domenici, Ranieri

AU - Consoloni, Lara

AU - Barbaro, Anna M.

AU - Pitacco, Paola

AU - Sorçaburu-Cigliero, Solange

AU - Corato, Silvia

AU - Moratti, Marco

AU - Fabbri, Matteo

AU - Schneider, Peter M.

AU - Salvaderi, Luca

AU - Fattorini, Paolo

AU - Giardina, Emiliano

AU - Trizzino, Lucia

AU - Plizza, Laura

AU - Turchi, Chiara

AU - Nicolin, Vanessa

AU - Grignani, Pierangela

AU - Vatta, Paolo

AU - Alù, Milena

AU - Baldassarra, Stefania Lonero

AU - Casarino, Lucia

AU - Previderè, Carlo

AU - Turrina, Stefania

AU - Pelotti, Susi

AU - Resta, Nicoletta

AU - Piccinini, Andrea

AU - Vecchiotti, Carla

AU - Ricci, Ugo

AU - Robino, Carlo

AU - De Stefano, Francesco

AU - Barbaro, Anna M.

AU - Carracedo, Angel

AU - Marrubini, Giorgio

PY - 2014

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AB - 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 (r2 = 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.

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