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- Insertion/Delection Polymorphism and forensic aplications: A preliminary studyPublication . Vieira Da Silva, ClĆ”udia; Matos, Sara; Amorim, AntĆ³nio; Afonso Costa, HeloĆsa; Morais, Paulo; Santos, Rodolfo; Espinheira, Rosa; Santos, J. CostaThe human genetic identification is usually based on the study of STR markers, robust and reliable for samples containing relatively small quantities of DNA. Recent advances in forensic genetics have focused on the development of genotyping assays using shorter amplicons, in order to improve the successful amplification of degraded samples. Single Nucleotide Polymorphisms (SNP) and Insertion/Deletion polymorphisms (INDEL), length polymorphisms created by insertions or deletions of one or more nucleotides in the genome, have considerable potential in this kind of forensic samples, usually present in identification casework, since they can combine desirable characteristics of both, STR and SNP. In this study, a set of 30 biallelic Deletion/Insertion polymorphisms (DIP or INDEL) distributed over 19 autosomes plus Amelogenin in a single multiplex PCR reaction was applied to 100 healthy and unrelated caucasian individuals. Statistical analysis revealed that the 30 biallelic markers can provide satisfactory levels of informativeness for forensic demands.
- Forensic genetic analysis of South Portuguese population with the six dye PowerplexĀ® Fusion 6CPublication . Vieira Da Silva, ClĆ”udia; Afonso Costa, HeloĆsa; Porto, Maria JoĆ£o; Cunha, E; Corte Real, F.; Amorim, AntĆ³nioAs an improvement in efficiency and in Human Discrimination Power, the new six dye multiplex kit PowerPlexĀ® Fusion 6C System, by Promega, available for human identification can co-amplify 27 loci, in a single reaction, have been introduced in the last years with great success. This kit allows the amplification and detection of autosomal loci included in the expanded Combined DNA Index System CODIS, plus the loci Penta D, PENTA E and SE33 as well as Amelogenin for gender determination. Furthermore, this kit includes three Y āSTRs (DYS391, DYS576 and DYS570), allowing allelic attribution in a total of 27 loci. This genetic markers extension satisfies not only CODIS but also European Standard Set recommendations. Thinking about continuous human migration movements, especially in a very cosmopolitan region like Lisbon and south of Portugal, and also, in keeping population studies and actualized STR databases we decided to update our previous studies. Our sample is composed of 600 unrelated individuals, from paternity testing with laboratory identity anonymised. DNA was extracted by Prep-n-go BufferTM(Thermo-Fisher Scientific). PCR amplification was performed with PowerPlexĀ® Fusion 6C System, according to manufacturerās guidelines. Fragment analysis was carried out in an Applied BiosystemsĀ® 3500 Genetic Analyser. Electrophoresis results were analysed with GeneMapperĀ® ID-X v1.4. Allele frequencies and population statistics, including Hardy-Weinberg equilibrium p-values from exact test probabilities and forensic parameters were calculated with adequate software. In conclusion, our population information was updated in order to apply most recent data in our casework weight of evidence.
- Population Genetic Data for F13A01, FES/FPS, F13B and LPL in the South Portuguese PopulationPublication . Vieira da Silva, ClĆ”udia; Amorim, AntĆ³nio; Afonso Costa, HeloĆsa; Espinheira, Rosa; Costa Santos, JorgeDNA parentage testing is currently performed using several highly polymorphic short tandem repeats (STRs). In our routine casework, we apply two validated STRs kits, in order to have results in the 13 codis loci plus D2S1338, D19S433, PENTA E, PENTA D, and Amelogenin. In complex and deficient paternity cases it is often necessary to increment the number of studied STRs. For this reason, we introduced in our laboratory GenePrintĀ® FFFL Multiplex kit, which can provide results in F13A1, FES/FPS, F13B, and LPL using the GenePrintĀ® FFFL System (Promega, USA) kit. In this study, we analyzed 150 unrelated and healthy individuals from the south Portugal population. Allele frequencies and statistical parameters were estimated with Arlequin 3.5.1.2. Paternity Statistics were calculated using software package PowerStats v12. The forensic efficiency values suggested that loci F13A01, FES/FPS, F13B, and LPL are discriminative and very useful to solve complex forensic casework, and should be added to the set of STRs loci routinely used in Forensic laboratories. In conclusion, an additional 4 loci dataset was established for the south Portuguese population, which can be used for both forensic casework and complex kinship testing
- InvestigaĆ§Ć£o de parentesco biolĆ³gico: a importĆ¢ncia de marcadores adicionais em casos de especial complexidadePublication . Rodrigues, Diogo; Vieira Da Silva, ClĆ”udia; Carvalho, MĆ³nica; Afonso Costa, HeloĆsa; Sampaio, Lisa; Cunha, E; Corte Real, F.; Amorim, AntĆ³nioA grande maioria das perĆcias de investigaĆ§Ć£o de parentesco biolĆ³gico realizadas pelo Instituto Nacional de Medicina Legal e CiĆŖncias Forenses (INMLCF) tĆŖm inicio com a ordem do Tribunal para realizaĆ§Ć£o da mesma. O mais frequente Ć© o Tribunal dar ordem para nos serem presentes, como intervenientes, um trio constituĆdo por um suposto pai, uma mĆ£e e um(a) filho(a), havendo, no entanto, variaƧƵes quanto ao nĆŗmero ou tipo de intervenientes, o que pode resultar em maior dificuldade em apresentar resultados com a robustez desejada. Em qualquer dos casos, genericamente, as conclusƵes possĆveis de um estudo de parentesco biolĆ³gico e mais concretamente de um estudo de paternidade sĆ£o a exclusĆ£o ou nĆ£o exclusĆ£o de paternidade relativamente ao suposto pai em estudo. A conclusĆ£o pela nĆ£o exclusĆ£o Ć© sempre acompanhada pela valorizaĆ§Ć£o estatĆstica dos resultados, designadamente atravĆ©s do cĆ”lculo e apresentaĆ§Ć£o do Ćndice de Parentesco (IP) e da Probabilidade de Parentesco (W). No caso de uma perĆcia de investigaĆ§Ć£o de parentesco em que nos seja presente unicamente um filho biolĆ³gico do suposto pai e um suposto filho biolĆ³gico, sem possibilidade de estudo das amostras das respetivas mĆ£es biolĆ³gicas nem da amostra biolĆ³gica do suposto pai, e nos Ć© pedido o estudo sobre a possibilidade de ambos serem filhos biolĆ³gicos do mesmo pai, a impossibilidade de exclusĆ£o da paternidade pode estar associada a valores calculados de IP que podem ser pouco robustos. O ensaio InvestigatorĀ® HDplex permite o estudo de marcadores genĆ©ticos adicionais aos habitualmente estudados na rotina pericial do INMLCF.
- The role of DNA concentrations in forensic casework results : regression models applicationPublication . Vieira Da Silva, ClĆ”udia; Amorim, AntĆ³nio; Afonso Costa, HeloĆsa; Porto, Maria JoĆ£o; Corte Real, F.; Antunes, MariliaIn forensic DNA typing, short tandem repeats (STRs) are the most frequently genotyped markers in order to distinguish between individuals and to relate them to a crime or to exonerate the innocent. In recent years, new controversies have arisen with the advent of more sensitive techniques, allowing profiles to be recovered from minimum amounts of DNA, hence, bringing challenges to weight of evidence evaluation for forensic DNA profiles obtained from low template DNA samples. Introduction of interpretation models, or even new weight of evidence software should be accompanied by a measure of uncertainty that is part of any biological analysis. Specially, due to stochastic effects, the reliability of the obtained profiles might differ between machinery, workflow and also PCR settings in use in different laboratories. In this work we try to understand the relation between Peak Area, DNA concentration and also size marker, using adequate regression models. Buccal swabs from 180 individuals, with unknown identity, were selected for this study. DNA was extracted with prep-n-goā¢ buffer and quantified using QuantifilerĀ® Trio DNA Quantification kit in a 7500 Real-Time PCR System (Applied Biosystems). STR amplification was performed with Powerplex Fusion 6C amplification kit (Promega). Amplified PCR products were separated and detected in an Applied BiosystemsĀ® 3500 Genetic Analyzer using manufacturerās conditions. Electrophoresis results were analysed with GeneMapperĀ® ID-X v1.4. Statistical analysis was performed with R Studio. Our results allow having an important overview about the relation between DNA concentrations, peak area, and size of the studied genetic markers.
- QUANTIFILERĀ®TRIO DNA method performance in a collection of ancient samplesPublication . Vieira- Silva, C.; Lopes, J.; Afonso Costa, H.; Ribeiro, T.; Porto, M.J.; Dias, M; Cunha, E; Amorim, A,During the past few years significant progress has been made in solving technical challenges associated with STR profiling including the ability to analyze degraded DNA and low amounts of DNA. The result of these changes is that useful STR profiles can now be obtained from previously untypeable forensic DNA samples. Analysis of DNA from ancient material represents an important role in molecular anthropology, although there are many limitations concerning low DNA quantity and/or degraded DNA, and/or PCR inhibitors. These factors can make it difficult to decide whether to continue with STR analysis, which STR panel to use and how much DNA to add to PCR reaction. With all these constraints, DNA quantification represents an important tool to decide which method will follow in order to improve workflow and have good results in less time-consuming. The QuantifilerĀ® Trio DNA method provides a quality index (QI) to detect the presence of degraded DNA along with PCR inhibitors.This guide allows the selection of the optimal short tandem repeat (STR) analysis chemistry (autosomal, or miniSTR) and streamlines the workflow while increasing downstream analysis success rates. In order to compare DNA quality from different extraction methods, samples from 46 exhumed Middle Ages individuals were extracted with modified phenol-chloroform method and also PrepFiler Express BTAā¢ method. DNA was quantified with QuantifilerĀ® Trio DNA Quantification in an Applied BiosystemsĀ® 7500 Real-Time PCR System. Results were analyzed and allow us to point QuantifilerĀ® Trio method as an important tool in pre-STR typing methods in ancient samples
- Forensic Genetics as a Tool for Peace and Justice: An Overview on DNA QuantificationPublication . Vieira Da Silva, ClĆ”udia; Afonso Costa, HeloĆsa; Costa Santos, J.; Espinheira, RosaIn Forensic Genetics, DNA analysis is performed to obtain a Short Tandem Repeat (STR) profile from an evidence sample, which is then compared with the victim and suspect(s) reference sample STR profile, to determine their contribution to that evidence sample. However, forensic biological samples can be present in low quantities and be exposed to different environmental insults leading to DNA degradation and contamination by inhibitor compounds. Thus, it is desirable for a forensic scientist to have useful information about the forensic sample quantity and quality prior to STR amplification. New methods in Forensic DNA analysis for detecting, preserving, and quantifying DNA, as well as its recovery from different biological materials are continually being developed. Real-Time PCR (RT-PCR) assays for DNA quantification, like the recent QuantifilerĀ® Duo DNA quantification kit (Applied Biosystems) proved to be very useful in forensic samples. Since many samples, mainly those resulting from sexual assault cases are often composed by unbalanced male/female DNA mixtures, the knew RT-PCR quantification assay, developed to quantify relative male/ female DNA ratio contributes not only to total DNA determination but also to ascertain the presence and quantity of enough male DNA in the sample. These results are important to guide the optimal STR analysis selection, such as autosomal STR, Y-STR, or mini-STR, increasing downstream analysis success rates. In this work we present real forensic casework where the DNA amount and quality were important to guide the selection of the appropriate STR amplification kit in order to increase the success of profiling in the first attempt, reducing the number of samples that need to be reprocessed and thereby decreasing the turn around time in a forensic laboratory.
- Y-Filer PlusĀ® genetic characterization of caucasian individuals from South PortugalPublication . Vieira da Silva, ClĆ”udia; Afonso Costa, HeloĆsa; ProenƧa, Marta; Ribeiro, Teresa; Porto, Maria JoĆ£o; Amorim, AntĆ³nioDue to their paternal inheritance, Y-STRs offers particular perspectives for identification and kinship analysis and are also a precious tool in sexual assault cases with relatively high amount of female DNA and also in mixtures from multiple male donors. Nonetheless their value, there are some limitations to their use in forensic investigations since their ability to discriminate between individuals is considerably lower than that of the autosomal STRs set, mainly in cases with close or distant patrilineal relatives.One of the most recently developed Y-STR kit, Y-Filer PlusĀ® (Life Technologies, Foster city, USA), allows forensic geneticists to study 27 Y-chromosomal loci. All the 16 markers included in the Y-FilerĀ® kit (Life Technologies, Foster city, USA), plus 9 additional markers: DYS576, DYS627, DYS460, DYS518, DYS570, DYS449, DYS481, DYF387S1 and DYS533, six of which (DYS576, DYS627, DYS518, DYS570, DYS449 and DYF387S1) are characterized as ārapidly mutatingā, and can differentiate between unrelated individuals and possibly between male relatives.Allelic frequencies were estimated with Arlequin v. 3.5. Gene and Haplotype diversities were estimated according to Nei formula. The discrimination capacity was also calculated by dividing the number of different haplotypes by the total number of individuals in the sample. The fraction of unique haplotypes was determined as the percent proportion of unique haplotypes. In conclusion, the recently introduced Y-Filer PlusĀ® system provides innovative discriminatory power for forensic application
- Variabilidade da quantidade de ADN em zaragatoas bucais āestudo preliminarPublication . Lucas, Isabel; Oliveira, Rita; Dourado, Catarina; Dario, Paulo; Reis, R.; Vieira Da Silva, ClĆ”udia; Amorim, AntĆ³nio; Afonso Costa, HeloĆsa; SimĆ£o, F.; Ribeiro, Teresa; Porto, Maria JoĆ£o; Costa Santos, JorgeOs laboratĆ³rios de GenĆ©tica Forense tĆŖm como objetivo a obtenĆ§Ć£o de perfis genĆ©ticos com vista Ć identificaĆ§Ć£o humana, para a resoluĆ§Ć£o de perĆcias do Ć¢mbito cĆvel e criminal. Com vista Ć obtenĆ§Ć£o dos perfis genĆ©ticos dos indivĆduos, Ć© necessĆ”rio efetuar colheitas de amostras biolĆ³gicas dos mesmos, denominadas por amostras de referĆŖncia. As mais comummente usadas sĆ£o as obtidas por descamaĆ§Ć£o do epitĆ©lio da mucosa bucal, com recurso ao uso de zaragatoas bucais. A variabilidade da quantidade de cĆ©lulas colhidas atravĆ©s deste procedimento, pode ser originada por diversos fatores. O objetivo deste estudo Ć© avaliar fatores que eventualmente possam contribuir para tal facto.
- Metodologias para preparaĆ§Ć£o e extraĆ§Ć£o de ADN de fragmentos humanos antigos: uma revisĆ£o preliminarPublication . Franco, Magda; Correia Dias, Helena; Balsa, Filipa; Serra, Armando; Afonso Costa, HeloĆsa; Nascimento, Rui; Corte Real, Francisco; CainĆ©, Laura; Amorim, AntĆ³nioO LaboratĆ³rio de ADN Antigo do ServiƧo de GenĆ©tica e Biologia Forenses da DelegaĆ§Ć£o do Centro do Instituto Nacional de Medicina Legal e CiĆŖncias Forenses (INMLCF) tem como objetivo, entre outros, analisar fragmentos humanos antigos (ossos e dentes). Pretende assim, atravĆ©s de anĆ”lises genĆ©ticas, contribuir para estudos relacionados com a evoluĆ§Ć£o de populaƧƵes antigas humanas, assim como a sua relaĆ§Ć£o com a populaĆ§Ć£o atual. A reduzida quantidade de ADN e o elevado nĆvel de degradaĆ§Ć£o sĆ£o dois dos desafios mais comuns quando se trabalha com ADN antigo (aDNA). Ć crucial diferenciar o ADN exĆ³geno do aDNA autĆŖntico, visto que estas amostras estĆ£o sujeitas a contaminaƧƵes ambientais e humanas. Tendo em conta a pouca qualidade esperada das amostras, serĆ” mais eficaz o estudo do ADN mitocondrial (mtDNA) do que do ADN nuclear, jĆ” que o mtDNA estĆ” presente nas cĆ©lulas num maior nĆŗmero de cĆ³pias e apresenta uma configuraĆ§Ć£o molecular circular fechada, que resulta numa maior proteĆ§Ć£o. Por outro lado, como este ADN Ć© herdado apenas por via materna, o seu estudo nĆ£o Ć© muito utilizado nas identificaƧƵes individuais, mas Ć© uma fonte importante de ADN para realizar estudos populacionais, sendo que nos permite obter informaƧƵes sobre as linhagens maternas dos indivĆduos. De acordo com a literatura, os mĆ©todos de prĆ©-processamento usados antes da extraĆ§Ć£o de ADN podem afetar a autenticidade e a quantidade de ADN obtido. VĆ”rias tĆ©cnicas de prĆ©-tratamento sĆ£o utilizadas atualmente, tais como, limpeza da superfĆcie do osso com lixĆvia, seguido de limpeza com Ć”gua e, por fim, com etanol. TambĆ©m Ć© utilizada luz UV para garantir que o ADN extraĆdo serĆ” autĆŖntico e nĆ£o de fontes exĆ³genas. Em anĆ”lises forenses, os dentes e ossos sĆ£o normalmente reduzidos a pĆ³ para a extraĆ§Ć£o de ADN. Tem sido tambĆ©m demonstrado que Ć© possĆvel obter bons resultados com um mĆ©todo diferente -"scrapping"-, sendo que esta tĆ©cnica preserva melhor a amostra. No entanto, Ć© necessĆ”rio avaliar a idade, o estado e o nĆvel de degradaĆ§Ć£o do osso/dente, sendo que amostras frĆ”geis podem ser quebradas facilmente, mesmo com este mĆ©todo. Na fase de extraĆ§Ć£o, existem vĆ”rios mĆ©todos que podem ser usados: mĆ©todos baseados em colunas (rĆ”pidos,mas produzem pouca quantidade de ADN); e mĆ©todos baseados em sĆlica, precipitaĆ§Ć£o e microfiltros (demorados e trabalhosos, mas produzem melhores resultados). TambĆ©m existe o mĆ©todo de fenol-clorofĆ³rmio (ainda de referĆŖncia e utilizado por vĆ”rios anos), que Ć© trabalhoso e envolve riscos para o operador. Em suma, Ć© importante fazer uma revisĆ£o dos mĆ©todos mais adequados para o tratamento e extraĆ§Ć£o destas amostras difĆceis, tendo em conta a manutenĆ§Ć£o da integridade das mesmas, para que possam ser utilizadas em investigaƧƵes futuras ou expostas em museus. Assim, esta revisĆ£o serve como um primeiro passo para a implementaĆ§Ć£o das metodologias mais adequadas para a obtenĆ§Ć£o de aDNA autĆŖntico de restos humanos antigos para estudos futuros no LaboratĆ³rio de ADN Antigo do INMLCF.