Fluorescent dyes
have become the preferred method of detection for nucleic acids in
Molecular Biology. Some common examples include automated
fluorescent DNA sequencing, fluorescent genotyping, and quantitative
target detection techniques (e.g., Molecular Beacons, etc.).
Fluorescent dyes absorb light at a characteristic wavelength and
re-emit light at a second lower energy, longer wavelength. The
wavelength (nm) where photon energy is most efficiently captured is
defined as the Absorbancemax. The
wavelength (nm) where light is most efficiently released is defined
as the Emissionmax. For more
information on Fluorescence Excitation and Emission,
click here.
The absorbance/emission properties of fluorescent dyes are
affected by their environment, including solvent, pH, and
conjugation to other macromolecules (e.g., nucleic acids). For
example, the fluorescent properties of fluorescein dramatically
change in low pH. Free fluorescein has a pKa of 6.4 and shifts to
the protonated form in low pH buffers, which is poorly fluorescent.
Therefore, all studies done using fluorescein should be done at pH
7.0 or higher.
Changes in physical properties of fluorescein that occur after
conjugation to DNA were studied in detail by Sjoback et
al.1. When attached to a
single-stranded oligo, the pKa of fluorescein shifts from 6.4 to 6.9
and the quantum yield decreases from 0.93 to 0.72. Further, the
absorption efficiency (extinction coefficient) of fluorescein
decreases by 1/3 after conjugation to DNA2. On the other hand, rhodamine does not
undergo a similar change in absorption efficiency following
conjugation. Unfortunately, this kind of comprehensive analysis is
not available for most dyes.
At IDT, we recently conducted a systematic study to assess the
impact that conjugation to DNA has on the Abmax and Emmax
of 9 of the most commonly used fluorescent dyes. Dyes were
conjugated to the 5'-end of a 12-mer oligonucleotide and HPLC
purified to ensure that no free dye was present. Spectra were
measured using a Photon Technology International (PTI) fluorometer
at 50 nM dye concentration in a buffer similar to that used in real
time PCR assays, containing 50 mM Tris pH 8.0, 50 mM KCl, and 5 mM
MgCl2.
Actual measured dye-oligo spectra are presented in the left
column; click on a spectra to see an enlarged higher resolution
image. For comparison, the Abmax and
Emmax provided by the dye
manufacturers are presented in the right column; all of these
measurements were collected using free dye in various solvent/pH
conditions. Note that unconjugated dyes are hydrophobic and are
poorly soluble in water, so many of the standard reference spectra
were measured in methanol.
