Due to release of the reporter, there is a detectable increase in fluorescence, which is monitored in real-time PCR experiments.

TLS Online TPP Program

#Id: 2565

#Unit 13. Methods in Biology

Molecular beacons consist of a stem (5–7 bp), and a loop (18–30 base pairs) and are labeled at the 5′ and 3′ ends by fluorophores and quenchers, respectively. 

TLS Online TPP Program

#Id: 4707

#Unit 13. Methods in Biology

From the four possible transitions (n!p*, p!p*, n!s*, s!s*), only two can be elicited with light from the UV/Vis spectrum for some biological molecules: n!p* and p!p*. The n!s* and s!s* transitions are

energetically not within the range of UV/Vis spectroscopy and require higher energies.

TLS Online TPP Program

#Id: 4708

#Unit 13. Methods in Biology

Molecular (sub-)structures responsible for interaction with electromagnetic radiation are called chromophores. 

TLS Online TPP Program

#Id: 4709

#Unit 13. Methods in Biology

In proteins, there are three types of chromophores relevant for UV/Vis spectroscopy:

• peptide bonds (amide bond);

• certain amino acid side chains (mainly tryptophan and tyrosine); and

• certain prosthetic groups and coenzymes (e.g. porphyrine groups such as in haem).

TLS Online TPP Program

#Id: 4710

#Unit 13. Methods in Biology

The presence of several conjugated double bonds in organic molecules results in an extended p-system of electrons which lowers the energy of the p* orbital through electron delocalisation.

TLS Online TPP Program

#Id: 4711

#Unit 13. Methods in Biology

Common colorimetric and UV absorption assays