Fluorescent markers for high-throughput DNA screening applications A primer-based fluorescence detection system offers an alternative especially suited for automating homogenous DNA assays. Sandra K. Randall, Jay Ji, Bita Nakhai, Nate Lawrence, and Mark Manak Most such tests employ some method of amplification in order to enable the detection of the smallest possible quantities of target DNA in clinical samples. Common amplification strategies include target amplification (nucleic acid sequence-based amplification, strand displacement amplification, PCR), probe-based amplification (cascade rolling-circle amplification), and signal amplification (branched DNA, Digene hybrid capture assay). Homogenous assay methods offer the advantage that they do not require the operator to perform manual separation of the amplified target by means of gel electrophoresis or other methods. Once setup is complete, target detection can be accomplished without additional manipulation of the sample. A major advance for PCR-based tests has been the development of homogenous assays using fluorescence detection. Such assays facilitate high throughput by monitoring the accumulation of fluorescence in a closed tube. Once the sample extract and reagents are combined, the tube is sealed and does not need to be opened again. This method minimizes the likelihood of false-positive results due to carryover contamination of the sample (a notable shortcoming of many nucleic acid amplificationbased detection systems), facilitates sample tracking, and significantly reduces hands-on processing time. PCR-based fluorescent homogenous assays can be monitored using either a labeled hybridization probe (TaqMan, Molecular Beacons) or a labeled PCR primer (Amplifluor). When used with an instrument capable of measuring fluorescence accumulation in real time, each of these methods offers high-sensitivity target detection and quantitative measurements over a wide dynamic range. In real-time analysis, a PCR reaction is performed for a set number of cycles, and the amount of fluorescence is monitored during each cycle. The accumulation of fluorescence correlates to the amount of PCR
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DNA sequencers are used to automate the DNA sequencing process.
Real-Time PCR Instruments
Real-Time PCR Instruments are machines that amplify and detect DNA. They combine the functions of thermal cyclers and fluorimeters, enabling the process of real-time PCR.
Microplates are plastic plates or cassettes that containing a number (typically 96 or 384) of small wells arranged in rows. Microplates are used to conduct a number of chemical reactions at the same time.
Lab-on-a-Chip (LOC) Devices
Lab-on-a-chip (LOC) devices are integrated semiconductors that serve as a laboratory for the testing and analysis of very small chemical and clinical samples.
Fluorometers measure the amount of fluorescent radiation produced by a sample exposed to monochromatic radiation.
Topics of Interest
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