RESUMO
Single primer amplification is shown to yield a DNA profile that is reproducible when based on the sequence content of the amplicons rather than on the pattern of length polymorphism. The sequence-based profile increases in reliability with increasing numbers of cycles of amplification. This process uses an arbitrarily chosen primer and a low initial annealing temperature in order to amplify sequences from the whole metagenome present in a sample that may contain only trace DNA, and a large number of cycles to select subsets of sequences based on variable amplification efficiency. Using arrays, we demonstrate the utility and limitations of this approach for profiling the large metagenomes typical of soils and the trace DNA present in drug seizures. We suggest that this type of profiling will be most effective once next-generation sequencing and advanced sequence analysis becomes routine.
Assuntos
Primers do DNA/química , DNA/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de DNA/métodos , DNA/química , DNA/metabolismo , Primers do DNA/metabolismo , Ciências Forenses , Humanos , Metagenoma , Técnicas de Amplificação de Ácido Nucleico/normas , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/normas , SoloRESUMO
This paper reports the results of a commission to develop a field deployable rapid short tandem repeat (STR)-based DNA profiling system to enable discrimination between tissues derived from a small number of individuals. Speed was achieved by truncation of sample preparation and field deployability by use of an Agilent 2100 Bioanalyser(TM). Human blood and tissues were stabbed with heated stainless steel wire and the resulting sample dehydrated with isopropanol prior to direct addition to a PCR. Choice of a polymerase tolerant of tissue residues and cycles of amplification appropriate for the amount of template expected yielded useful profiles with a custom-designed quintuplex primer set suitable for use with the Bioanalyser(TM). Samples stored on wires remained amplifiable for months, allowing their transportation unrefrigerated from remote locations to a laboratory for analysis using AmpFlSTR(®) Profiler Plus(®) without further processing. The field system meets the requirements for discrimination of samples from small sets and retains access to full STR profiling when required.
Assuntos
Impressões Digitais de DNA/métodos , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Primers do DNA , Eletroforese Capilar , Genótipo , Temperatura Alta , Humanos , Repetições de Microssatélites , Reação em Cadeia da Polimerase , Aço InoxidávelRESUMO
The human sex test in forensic multiplexes is based on the amelogenin gene on both the X and Y chromosomes commonly used in sex genotyping. In this study of 338 male individuals in a Malaysian population comprising Malays, Chinese and Indians, using the AmpFlSTR Profiler Plus kit, the amelogenin test gave a significant proportion of null alleles in the Indian ethnic group (3.6% frequency) and 0.88% frequency in the Malay ethnic group due to a deletion of the gene on the Y chromosome. This sex test also failed in a forensic casework sample. Failure of the amelogenin test highlights the need for more reliable sex determination than is offered by the amelogenin locus in the Malay and Indian populations. The gender of the Indian-Malay amelogenin nulls was confirmed by the presence of three Y-STR alleles (DYS438, DYS390 and DYS439). For the Indian ethnic group, one of the Y-STR forms a stable haplotype with the amelogenin null. The amelogenin-deletion individuals also showed a null with a male-specific minisatellite MSY1, indicating that a very large deletion was involved that included the amelogenin and the MSY1 loci on the short arm of the Y chromosomes (Yp).
Assuntos
Impressões Digitais de DNA/métodos , Proteínas do Esmalte Dentário/genética , Etnicidade/genética , Genética Populacional , Processos de Determinação Sexual , Alelos , Amelogenina , China/etnologia , Cromossomos Humanos Y , Deleção de Genes , Humanos , Índia/etnologia , Malásia/etnologia , Masculino , Sequências de Repetição em Tandem , Germe de DenteRESUMO
Current procedures for human DNA quantitation reach their limit at 150 pg DNA, which is above the limit of the PCR profiling range using Profiler-Plus (Applied Biosystems, CA). This study tested the potential for the use of primate specific Alu sequences in forensic science for the sensitive detection and quantitaion of DNA. A fluorescently labelled primer pair was designed enabling high efficiency amplification of the core Alu sequence within primate DNA. Quantitation was performed by measurement of fluorescence intensity and comparison to a series of standard template DNA amounts via the construction of a standard curve. The new Alu-based quantitation protocol developed has shown its feasibility in more sensitively quantitating (100-2.5 pg) unknown amounts of human DNA for forensic use. The method is compatible with the use and throughput of current forensic procedures.