DNA microarray is a high-throughput approach used for analyzing complex nucleic samples that have limited feasible availability due to the complicated procedure and expensive consumption. Nonetheless, their throughput is low because of the limited number of channels in the real-time system. Real-time quantitative PCRs have been widely used to quantify target genes with high sensitivity, specificity and a wide dynamic range 13, 14, 15, 16. Indeed, several multiplex detection methods of target genes have been developed and employed in the fields of food/feed identifications 1, 2, 3, 4, medical diagnostics 5, 6, 7, and large scale sequencing 8, 9, 10.Īlthough conventional multiplex PCRs have always been used for amplification of nucleic acid samples, problems such as preferential amplification of shorter DNA templates, interference of multiple primer pairs and limited substrates limit their wide use in quantitative and high-throughput research 11, 12. Therefore, it is of great importance to develop an economical and more effective technique that would enable simultaneous qualitative and quantitative detection of these samples. When dealing with complex nucleic acid samples, multiple reactions need to be performed separately, which is costly and time consuming. Traditional nucleic acid based technologies can usually detect only one target gene in one reaction. The increases in the number of biomolecular samples that need to be analyzed generate a great demand for a high-throughput detection method. To sum up, the new EPFS system is the first analytical technology of this kind that enables simultaneous qualitative, quantitative and high-throughput analysis of multiple genes. Consequently, the same qualitative, quantitative and high-throughput results were confirmed with the four GM maize. Moreover, the reproducibility assays were further performed using four foodborne pathogenic bacteria to further evaluate the applicability of the system. In addition, the quantitative results revealed that the absolute limit of detection was 10 3 copies, showing good repeatability. The qualitative results revealed high specificity and sensitivity of 0.5% (w/w). The sensitivity and specificity of the system was examined using four kinds of genetically modified (GM) maize. After product purification, different fluorescent-labeled DNA products were qualitatively analyzed by the fluorescent intensity determination. Through emulsion PCR, a target DNA was amplified in droplets that functioned as micro-reactors. In a single reaction set, each pair of primers was labeled with a specific fluorophore. Biotechniques 30, 264–266 (2001).We constructed and validated a novel emulsion PCR method combined with fluorescence spectrophotometry (EPFS) for simultaneous qualitative, quantitative and high-throughput detection of multiple DNA targets. Running gels backwards to select DNA molecules larger than a minimum size. Accurate multiplex polony sequencing of an evolved bacterial genome. Genome sequencing in microfabricated high-density picolitre reactors. PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Molecular haplotyping by linking emulsion PCR: analysis of paraoxonase 1 haplotypes and phenotypes. Single-molecule reverse transcription polymerase chain reaction using water-in-oil emulsion. Generic expansion of the substrate spectrum of a DNA polymerase by directed evolution. Directed evolution of polymerase function by compartmentalized self-replication. Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning. Bias in template-to-product ratios in multitemplate PCR.
0 Comments
Leave a Reply. |