PCR primers were developed to amplify an 250 bp region surro

PCR primers were developed to amplify an ~250 bp region surrounding the BRAF V600E region in exon 15. Amplification was detected using adjacent dual hybridization fluorescence resonance energy transfer (FRET) probes. The donor probe was 24 nucleotides long and a 100% match with the wild type sequence between nucleotides 1791 and 1814 and labeled at its 3\' end with the blue donor molecule carboxyfluoroscein. The donor probe was 29 nucleotides long, labeled at its 5\' end with the fluorophore LC-Red640, and was a perfect match to the wild-type BRAF sequence between nucleotides 1816 and 1844. Extracted DNA was mixed with primers and probes and amplified in the Roche LightCycler 480 with real-time detection of amplification product during the annealing phase of each cycle. Explain the principle of FRET.

Solution

FRET analysis is a distance based radiationless tool available for measuring nanometer scale distance and the change in distance both in vivo and in vitro. Due to its sensitiivity, it is used to detect molecular level of interaction, FRET can yield a significant amount of structural information.it inolve transfer of excited state energy from one flurophore to another. In the first step, absorption of energy by the donar molecule, resulting in excitation from the ground state. If suitable acceptor flurophore is nearby, then nonradiative energy transfer between donar and acceptor can occur. This transfer involves resonance between the singlet singlet electronic transition of the two flurophores, generated by coupling of the emissiontransition dipol moment of the donar and the absorption transition dipol moment of the acceptor. First member of FRET pair function as an energy as a donar and second member of FRET pair function as an acceptor.