The molecular characterisation of a depurinated trial dna sample can be a model to understand the reliability of the results in forensic genetics.

Gregorio Seidita, Chiara Turchi, Vanessa Nicolin, Pierangela 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 MarrubiniAndrea Verzeletti, Eugenia Carnevali, Francesca Scarnicci, Ranieri Domenici, Lara Consoloni, Paola Pitacco, Solange Sorçaburu-Cigliero, Silvia Corato, Marco Moratti, Matteo Fabbri, Peter M. Schneider, Luca Salvaderi, Paolo Fattorini, Emiliano Giardina, Lucia Trizzino, Laura Plizza

    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 post mortem 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 out 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. This data shows 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 characterise 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 characterised 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 4,500 amplicons, the frequency of PCR artefacts (allele drop out, 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)-
    Numero di pagine0
    RivistaElectrophoresis
    Volume00
    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
    • Clinical Biochemistry
    • Biochemistry

    Cita questo

    The molecular characterisation of a depurinated trial dna sample can be a model to understand the reliability of the results in forensic genetics. / Seidita, Gregorio; 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; Verzeletti, Andrea; Carnevali, Eugenia; Scarnicci, Francesca; Domenici, Ranieri; Consoloni, Lara; Pitacco, Paola; Sorçaburu-Cigliero, Solange; Corato, Silvia; Moratti, Marco; Fabbri, Matteo; Schneider, Peter M.; Salvaderi, Luca; Fattorini, Paolo; Giardina, Emiliano; Trizzino, Lucia; Plizza, Laura.

    In: Electrophoresis, Vol. 00, 2014, pag. -.

    Risultato della ricerca: Article

    Seidita, G, 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, Verzeletti, A, Carnevali, E, Scarnicci, F, Domenici, R, Consoloni, L, Pitacco, P, Sorçaburu-Cigliero, S, Corato, S, Moratti, M, Fabbri, M, Schneider, PM, Salvaderi, L, Fattorini, P, Giardina, E, Trizzino, L & Plizza, L 2014, 'The molecular characterisation of a depurinated trial dna sample can be a model to understand the reliability of the results in forensic genetics.', Electrophoresis, vol. 00, pagg. -.
    Seidita, Gregorio ; 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 ; Verzeletti, Andrea ; Carnevali, Eugenia ; Scarnicci, Francesca ; Domenici, Ranieri ; Consoloni, Lara ; Pitacco, Paola ; Sorçaburu-Cigliero, Solange ; Corato, Silvia ; Moratti, Marco ; Fabbri, Matteo ; Schneider, Peter M. ; Salvaderi, Luca ; Fattorini, Paolo ; Giardina, Emiliano ; Trizzino, Lucia ; Plizza, Laura. / The molecular characterisation of a depurinated trial dna sample can be a model to understand the reliability of the results in forensic genetics. In: Electrophoresis. 2014 ; Vol. 00. pagg. -.
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    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 post mortem 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 out 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. This data shows 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 characterise 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 characterised 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 4,500 amplicons, the frequency of PCR artefacts (allele drop out, 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.",
    author = "Gregorio Seidita and Chiara Turchi and Vanessa Nicolin and Pierangela Grignani and Paolo Vatta and Milena Al{\`u} and Baldassarra, {Stefania Lonero} and Lucia Casarino and Carlo Previder{\`e} and Stefania Turrina and Susi Pelotti and Nicoletta Resta and Andrea Piccinini and Carla Vecchiotti and Ugo Ricci and Carlo Robino and {De Stefano}, Francesco and Barbaro, {Anna M.} and Angel Carracedo and Giorgio Marrubini and Andrea Verzeletti and Eugenia Carnevali and Francesca Scarnicci and Ranieri Domenici and Lara Consoloni and Paola Pitacco and Solange Sor{\cc}aburu-Cigliero and Silvia Corato and Marco Moratti and Matteo Fabbri and Schneider, {Peter M.} and Luca Salvaderi and Paolo Fattorini and Emiliano Giardina and Lucia Trizzino and Laura Plizza",
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    AU - Seidita, Gregorio

    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

    AU - Verzeletti, Andrea

    AU - Carnevali, Eugenia

    AU - Scarnicci, Francesca

    AU - Domenici, Ranieri

    AU - Consoloni, Lara

    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

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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 post mortem 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 out 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. This data shows 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 characterise 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 characterised 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 4,500 amplicons, the frequency of PCR artefacts (allele drop out, 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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