RESUMO
We describe a highly discriminating multiplex short tandem repeat PCR human identification system that gives a matching probability for Caucasians of European ancestry of 2.94 x 10(-8) or 5.66 x 10(-10) when used in combination with a previously described system. The system produces discrimination equal to or greater than four single locus probes (restriction fragment length polymorphism [RFLP] typing of variable nucleotide tandem repeat [VNTR] loci). The test is robust and reproducible and works with 1-10 ng of template DNA, using fluorescent detection of PCR products from either 4 or 6 short tandem repeat loci and the X-Y homologous gene amelogenin, giving simultaneous sex diagnosis.
Assuntos
Medicina Legal , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Ácido Nucleico , Análise para Determinação do Sexo , Alelos , Sequência de Bases , Mapeamento Cromossômico , Humanos , Dados de Sequência MolecularAssuntos
DNA/análise , Fluorometria/instrumentação , Análise de Sequência de DNA/instrumentação , Automação , Primers do DNA , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Desoxirribonucleotídeos/química , Eletroforese em Gel de Poliacrilamida/instrumentação , Eletroforese em Gel de Poliacrilamida/métodos , Desenho de Equipamento , Falha de Equipamento , Escherichia coli/genética , Corantes Fluorescentes/análise , Fluorometria/métodos , Indicadores e Reagentes , Resinas de Troca Iônica , Desnaturação de Ácido Nucleico , Mutação Puntual , Reação em Cadeia da Polimerase , Análise de Sequência de DNA/métodos , Dióxido de Silício , Software , Manejo de Espécimes , Taq Polimerase/genética , Taq Polimerase/metabolismo , Moldes Genéticos , Ureia/farmacologiaRESUMO
Through the use of fluorescence-based polymerase chain reaction systems, a highly discriminating multiplex with the potential for individual identification has been developed. The use of multiple dye technology enabling loci with overlapping size ranges to be co-amplified has enabled us to successfully amplify seven tetranucleotide short tandem repeat loci within a single reaction resulting in a discriminating power in the region of 1 x 10(9). Three out of the seven loci employed exhibit alleles differing in size by only 2 bp as opposed to the conventional 4 bp, which results in such loci being more powerful in terms of distinguishing between samples, particularly when co-amplified in this manner. The size ranges of the loci contained within the system are such that windows still exist for the inclusion of additional loci at a later stage, which could increase the discriminating power of the system still further. In addition, further weight and utility is lent to the system through the incorporation of a simple and reliable sex test involving the amplification of a segment of the X-Y homologous gene Amelogenin.
Assuntos
DNA/análise , Proteínas do Esmalte Dentário/genética , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Amelogenina , Sequência de Bases , DNA/química , Feminino , Corantes Fluorescentes , Humanos , Masculino , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Germe de DenteRESUMO
Short tandem repeat (STR) loci are routinely employed for individual identification. WE have examined the performance and reproducibility of a highly informative co-amplification system containing the tetranucleotide STR loci: HUMVWFA31/A, HUMTH01, D20S85, D8S1179, HUMFIBRA, D21S11, and D18S51, in conjunction with the amelogenin sex test, in addition to a modified system omitting the locus D20S85. Polymerase chain reaction (PCR) products were fluorescently detected on an automated sequencer and automatically sized against an internal size standard by Genescan software. Both systems were routinely able to type 500 pg of undegraded DNA. At DNA concentrations between 50-500 pg, partial profiles were produced, but no allelic drop-out was observed. Balanced amplification of all loci occurred over a wide range of DNA concentrations from 50 pg to 10 ng. Alteration of reagent concentrations and cycling parameters from optimal resulted in variation in the efficiency of individual locus amplification relative to the other loci within the system. This was also observed at high ionic strength or extreme pH. However, at all reagent concentrations and conditions, allelic drop-out was not observed. These multiplex systems have potential in both routine forensic and intelligence database applications.
Assuntos
Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Ácido Nucleico , Soluções Tampão , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Concentração Osmolar , Reprodutibilidade dos Testes , Taq Polimerase , TemperaturaRESUMO
The Applied Biosystems (ABI) Prism 377 DNA sequencer has been evaluated in an attempt to increase the throughput of samples for short tandem repeat (STR) analysis, in both forensic casework and the UK National Criminal Intelligence DNA Database. The gel system assessed consisted of 0.2 mm, 4% acrylamide 6 M urea gels, with a well-to-read distance of 36 cm. Gels were run at a constant voltage of 3 kV and constant temperature of 51 degrees C. The run time of our second generation multiplex (SGM) STR system was achieved in less than 2 h. Rigorous validation has been performed on the instrument hardware and software. Complete resolution of 1 base differences was obtained, up to and beyond 350 bases; sizing precision across gels was more than 2-fold higher than the 373A and the sensitivity was increased by one third.