Multiplex Realtime Polymerase Chain Reaction Methodology

Multiplex PCR is the amplification of multiple targets, or the capability of a system to amplify multiple targets, within the same reaction vessel. ''Real time'' refers to the fact that the detection of the amplified product is taking place in real time or during the actual process of the amplification reaction. Therefore multiplex real-time PCR

involves the simultaneous amplification, detection, and discrimination of multiple amplified targets in a single reaction. Real-time PCR and multiplexing methodologies, when combined, provide a flexible research or clinical tool that can be specifically tailored to interrogate nucleic acid sequences and to provide a researcher with experimental answers to a variety of questions. Flexibility is a product of the variety of chemistries available to perform real-time PCR, in conjunction with the numerous strategies available to combine reactions in a multiplexed fashion.

The detection of real-time PCR products is based on increases in fluorescence that are apparent only when amplified products are present. Real-time PCR instrumentation uses a light source to produce an excitation wavelength that activates fluorescent molecules within the reaction. Following the interaction between the fluorescent molecule or probe and the target amplicon, fluorescent emissions can be observed and monitored. The specific method by which products in a reaction are discriminated depends on the particular detection chemistry being utilized. Some of the most commonly utilized chemistries or fluorescence detection schemes for realtime PCR detection include: 1) 5' nuclease or ''TaqMan'' probes;[1] 2) hybridization probes or fluorescence resonance energy transfer (FRET) probes;[2] 3) molecular beacons;[3] 4) minor groove binding probes;[4] and 5) double-stranded DNA binding dyes (SYBR green).[5]

Each different detection chemistry has specific characteristics that lend to different multiplexing strategies. Multiplexing is generally achieved by using multiple primer and/or probe sets in a reaction, although there may also be cases where a single primer set is used with multiple probes, or a single probe could be used with a number of different primer sets. The amplified products generated can be discriminated by means of melting curve analysis,[6] by an analysis and discrimination of fluorescent emissions,[7] or by a combination of the two. When the different strategies are combined with the variety of available chemistries, a vast number of permutations and possibilities are created. The strategy utilized will depend on the information desired by the researcher.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment