A major limitation of FRET is:
FRET efficiency depends on spectral overlap integral between donor emission spectrum and acceptor absorption spectrum, high donor quantum yield, acceptor extinction coefficient, and favorable dipole orientation. If emission spectra overlap excessively, direct acceptor excitation and bleed-through degrade signal. Non-optimal separation complicates filter sets and requires extensive corrections or spectral unmixing. Additionally, photobleaching and environmental sensitivity affect quantification. Unlike radioactivity or heat which are not intrinsic, requirement for precise spectral matching and
Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.
