RESUMO
We demonstrate a conduit for the delivery of a step change in the DNA analysis process: A fully integrated instrument for the analysis of multiplex short tandem repeat DNA profiles from reference buccal samples is described and is suitable for the processing of such samples within a forensic environment such as a police custody suite or booking office. The instrument is loaded with a DNA processing cartridge which incorporates on-board pumps and valves which direct the delivery of sample and reagents to the various reaction chambers to allow DNA purification, amplification of the DNA by PCR, and collection of the amplified product for delivery to an integral CE chip. The fluorescently labeled product is separated using micro capillary electrophoresis with a resolution of 1.2 base pairs (bp) allowing laser induced fluorescence-based detection of the amplified short tandem repeat fragments and subsequent analysis of data to produce a DNA profile which is compatible with the data format of the UK DNA database. The entire process from taking the sample from a suspect, to database compatible DNA profile production can currently be achieved in less than 4 h. By integrating such an instrument and microfluidic cartridge with the forensic process, we believe it will be possible in the near future to process a DNA sample taken from an individual in police custody and compare the profile with the DNA profiles held on a DNA Database in as little as 3 h.
Assuntos
DNA/análise , Genética Forense/métodos , Técnicas Analíticas Microfluídicas/métodos , Bases de Dados de Ácidos Nucleicos , Reação em Cadeia da Polimerase , Manejo de Espécimes , Fatores de TempoRESUMO
The microfluidic integration of an entire DNA analysis workflow on a fully integrated miniaturized instrument is reported using lab-on-a-chip automation to perform DNA fingerprinting compatible with CODIS standard relevant to the forensic community. The instrument aims to improve the cost, duration, and ease of use to perform a "sample-to-profile" analysis with no need for human intervention. The present publication describes the operation of the three major components of the system: the electronic control components, the microfluidic cartridge and CE microchip, and the optical excitation/detection module. Experimental details are given to characterize the level of performance, stability, reliability, accuracy, and sensitivity of the prototype system. A typical temperature profile from a PCR amplification process and an electropherogram of a commercial size standard (GeneScan 500™, Applied Biosystems) separation are shown to assess the relevance of the instrument to forensic applications. Finally, we present a profile from an automated integrated run where lysed cells from a buccal swab were introduced in the system and no further human intervention was required to complete the analysis.
Assuntos
Eletroforese Capilar/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Repetições de Microssatélites , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/instrumentação , Bochecha , DNA/química , DNA/isolamento & purificação , Genética Forense/métodos , Humanos , Técnicas Analíticas Microfluídicas/métodos , Mucosa Bucal/citologia , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodos , TemperaturaRESUMO
A novel anthracene-tagged oligonucleotide can discriminate between a fully-matched DNA target sequence and one with a single mismatching base-pair through a remarkable difference in fluorescence emission intensity upon duplex formation.
Assuntos
Antracenos/química , Pareamento Incorreto de Bases/genética , DNA/genética , Corantes Fluorescentes/química , Sondas de Oligonucleotídeos/química , DNA/química , Corantes Fluorescentes/síntese química , Estrutura Molecular , Sondas de Oligonucleotídeos/síntese química , Polimorfismo de Nucleotídeo Único/genética , Espectrometria de Fluorescência , EstereoisomerismoRESUMO
The ground- and excited-state properties of a series of photochromic barbiturate receptors (N,N'-bis{6-[omega-(anthracen-9-yloxy)alkanoylamino]pyridin-2-yl}-5-t-butyl-isophthalamide, Tn), in which anthracene chromophores are tethered via (CH2)n (n = 1, 3-6) polymethylene linkers to the H-bond receptor moiety, are described. In these systems, the thermally reversible [4pi + 4pi] photodimerization of the anthracenes yields macrocyclic receptors (TnC) that possess significantly reduced affinity toward barbital as compared to their acyclic counterparts. The length of the tether not only determines the overall binding ability of the cyclized receptor but also has a profound influence on the photochemical and photophysical properties of the anthracene chromophores. The reduced mobility experienced by the covalently bound anthracenes controls the reactivity of a fluorescent excimer that is proposed to be an intermediate in the photocyclization process.
RESUMO
A heterodifunctionalised ferrocene, containing a carboxylic acid and an amidopyridine unit, self-assembles in organic solvents through complementary hydrogen bonds.