Fret Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Fret and links to relevant pages within the course.

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Human Papillomavirus (HPV) and Molecular Diagnostic Testing
Cervista HPV DNA Cervista HPV (Third Wave Technologies, Hologic, Inc, Madison, WI)Two assays are available:1. Cervista HPV HR Screening Test14 HR-HPV Types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, or 682. Cervista HPV 16/18Identifies and differentiates HR Types 16 and 18, referred to as HPV-16/18 genotyping testIn the Cervista HPV DNA assays, two hybridizations and an enzyme cleavage occur in a primary reaction. The secondary reaction is detection with signal amplification using fluorescence resonance energy transfer (FRET). Prior to hybridization, the HPV DNA (if present) is isolated, denatured, and added to microplate wells.	View Page
Cervista HPV DNA Secondary Reaction In the secondary reaction, the cleaved 5' Flaps attach to a FRET oligonucleotide probe. The FRET probe is curved such that the 5' Flap end nucleotide base pair attaches to the FRET probe in two places. Another cleavage reaction cleaves the 5' Flaps off the FRET probes and excites the fluorophores. The 5' Flaps reanneal and a cycling of the 5' Flaps off and on the FRET probes occurs. The emitted light is detected in a fluorescence plate reader. Because of the increasing number of 5' Flaps created in the primary reaction and the cycling of the 5' Flaps off and on the FRET probes, the target-specific signal is consequently amplified. During a 4-hour testing period, on average, the two reactions result in a 1-10 million-fold signal amplification.	View Page

Real-Time PCR
Detection Methods There are two main methods for detection of the real-time PCR products: non-specific fluorescent dyes and sequence-specific DNA probes.Non-specific dyesThese are dyes that bind to all pieces of double-stranded DNA and result in fluorescence. SYBR green is an example of a non-specific dye that is commonly used in real-time PCR. Because these dyes bind to all double-stranded DNA sequences, the fluorescence intensity increases after each cycle and allows for the concentrations to be determined. Since there is no associated unit of measure, only a fraction or ratio to a standard dilution can be determined. One problem with this method is that the dyes are non-specific and will even bind with primer dimers. This can potentially cause inaccurate quantification of the intended target sequence. However, if the specificity is not required, this is a more cost-effective method than the sequence-specific DNA probe method. Sequence-specific DNA probesAs the name implies, this method of detection is more specific than non-specific dyes. Probes can be designed to bind only to certain DNA sequences and are labeled with a fluorescence reporter that permits detection only after hybridization. The use of these sequence-specific probes allows for the detection of only the specific DNA product and utilizes fluorescence resonance energy transfer (FRET). These probes are also used for multiplexing. Multiplexing is the process of assaying several different genes in the same reaction. In this process, each probe has a different color reporter, which allows for the quantification of several different sequence products at the same time.	View Page
FRET Fluorescence resonance energy transfer (FRET) is the process that permits probes to emit fluorescence only after hybridization and describes the energy transfer between two fluorophores. Each probe consists of both a donor and a receptor fluorophore with the donor being on the 3' end and the reporter on the 5' end. The proximity of the two fluorophores to each other allows for the donor to reduce the fluorescent state of the reporter. When the probe attaches to the single-stranded piece of DNA, the fluorescence is still being suppressed. As the polymerase takes effect and begins to synthesize the new complementary strand of DNA, the reporter and donor fluorophores are separated. This separation allows for the reporter to emit its energy, which is picked up by the computer. The more denaturing and annealing that takes place, the higher the fluorescence in the tube. The process of FRET can be compared to the classic science experiment using tuning forks. If you strike one tuning fork so that it rings and hold it near a tuning fork that has not been struck, it will begin to ring by the force of the resonating waves from the initial tuning fork.	View Page
In real-time PCR procedures, fluorescence resonance energy transfer (FRET) is the process that allows for probes to emit fluorescence only after hybridization.	View Page

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