RESUMO
We developed an integrated chip for real-time amplification and detection of nucleic acid using pH-sensing complementary metal-oxide semiconductor (CMOS) technology. Here we show an amplification-coupled detection method for directly measuring released hydrogen ions during nucleotide incorporation rather than relying on indirect measurements such as fluorescent dyes. This is a label-free, non-optical, real-time method for detecting and quantifying target sequences by monitoring pH signatures of native amplification chemistries. The chip has ion-sensitive field effect transistor (ISFET) sensors, temperature sensors, resistive heating, signal processing and control circuitry all integrated to create a full system-on-chip platform. We evaluated the platform using two amplification strategies: PCR and isothermal amplification. Using this platform, we genotyped and discriminated unique single-nucleotide polymorphism (SNP) variants of the cytochrome P450 family from crude human saliva. We anticipate this semiconductor technology will enable the creation of devices for cost-effective, portable and scalable real-time nucleic acid analysis.
Assuntos
Concentração de Íons de Hidrogênio , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Semicondutores , Análise de Sequência de DNA/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Desenho de Equipamento , Integração de SistemasRESUMO
The Brassicaceae are targets for DNA manipulation to modify oil content and composition. However, any strategy for creating novel products using genetic modification or traditional breeding must take into account the potential for hybridization with other Brassica species, many of which are important sources of edible oils. In this study we have tested Brassica carinata, a possible target for oil modification, to establish whether it can cross with other Brassica species and related genera, and we have developed molecular DNA assays to confirm hybridization.
Assuntos
Brassica/genética , Brassicaceae/genética , Quimera/genética , Testes Genéticos/métodos , RNA Ribossômico 5S/genética , Cruzamentos Genéticos , DNA Ribossômico/análise , DNA Ribossômico/genética , Marcadores Genéticos/fisiologia , Genoma de Planta/genética , Filogenia , Plantas Geneticamente Modificadas/genética , Raphanus/genética , Sinapis/genética , Especificidade da EspécieRESUMO
DNA extractions are a major cost for high-throughput genotyping. The loop-mediated isothermal amplification (LAMP) assay has been used for the detection of two genetically modified (GM) related sequences. The amplification of target DNA sequences from leaf and maize seed tissues prepared with minimum preparative treatment (disruption in water) demonstrates the ability of LAMP to work in conditions normally inhibitive to PCRs. The wide dynamic range of detection in these samples suggests that LAMP is highly sensitive even when the target is presented in such a crude form. LAMP offers a means of reducing genotyping costs as well as simplifying testing procedures.
Assuntos
DNA de Plantas/genética , Ensaios de Triagem em Larga Escala/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Zea mays/genética , Genótipo , Técnicas de Amplificação de Ácido Nucleico/economiaRESUMO
Estimation of the genetic relatedness of traditional olive cultivars with genetic markers and phenotypic data enables progress in plant breeding, management of genetic resources, and protection of both breeders' rights and certified premium products. We used amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), and quantitative and qualitative morphological traits, including characteristics recommended for variety registration, to study genetic diversity and relationships in the olive at different levels. The 14 varieties analyzed, which are used for the production of Protected Denomination of Origin extra-virgin olive oil, represent the most important cultivars in the Campania region of Italy and typify a regional diversity characteristic of traditional olive cultivation. The genetic distances obtained with the two DNA marker systems were significantly correlated, as were those obtained by quantitative and qualitative traits. A lower but significant correlation was also observed between distances based on molecular markers and quantitative traits, but qualitative traits, even if sampled in high numbers, failed to describe the pattern of molecular similarity. Our data imply that the type and the number of phenotypic traits scored can greatly influence the outcome of the analysis, and care should be taken when qualitative and quantitative data are combined. Furthermore, the data indicate that the two molecular marker systems are useful for investigating genetic relationships, but they may also be used to complement and assist the traditional registration of varieties. We propose that since the information provided by molecular and morphological marker systems in olive is different, they should serve different purposes.
Assuntos
Repetições de Microssatélites/genética , Olea/classificação , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Marcadores Genéticos , Variação Genética , Genótipo , Itália , Olea/genética , Fenótipo , Filogenia , Polimorfismo GenéticoRESUMO
BACKGROUND: The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR). Here we have applied the loop-mediated isothermal amplification (LAMP) method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene) junctions. RESULTS: We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. CONCLUSION: This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.