Spectrophotometers
Nucleic Acids and Proteins
Nucleic Acids and
Spectrophotometer
• The rings of the bases (A, C, G, T, U)
are made up of alternating single and
double bonds.
• Such ring structures absorb in the U.V.
• Each of the four nucleotide bases has a
slightly different absorption spectrum,
and the spectrum of DNA is the average
of them.
Molar Extinction
Coefficients of Bases
Adenine
 (Epsilon)
(molar extinction
coefficient) at OD260
15,200
Cytosine
7,050
Guanine
12,010
Thymine
8,400
Base
DNA and
Spectrophotometers
• Double stranded DNA is a dynamic
structure and not a static entity.
• The two strands are held together by
non-covalent interactions (hydrogen
bonding and base stacking).
• The energy of these interactions
allows the helix to come apart quite
easily at physiological temperatures.
Double-Stranded vs.
Single-Stranded Nucleic
Acids
• DNA can be heated and, at a certain
temperature, the two strands will come
apart. We say that the DNA helix has
melted or denatured.
• This transition can be followed by the
increase in the absorption of ultraviolet
light by the molecule as it goes from helix
to random coil (the denatured form). This
is called hyperchromicity:
Bases Are Exposed in SS
DNA
• When a DNA helix is denatured to
become single strands the absorbance is
increased about 30 percent.
• This increase, (the hyperchromic shift)
indicates that the double-stranded
molecule is quenching fluorescence.
• So, you always need to know if your DNA
is double or single stranded when
measuring it using the
spectrophotometer.
UV radiation can be used to sterilize: the absorbed
energy destroys the DNA and kills the organism.
Spectrophotometer and
Nucleic Acid
Applications
• Measure the complexity of double
stranded DNA (COT analysis)
• Measure the concentration of double
stranded or single stranded DNAs,
nucleotide mixes and RNA in
solution.
• Measure how clean the DNA/RNA is
relative to contaminating protein.
Molar Extinction
Coefficient
• For solution concentrations given in
mol/liter and a cuvette of 1-cm path
length, E is the molar extinction
coefficient and has units of
M-1cm-1.
• If concentration units of ug/ml are used,
then E is the specific absorption
coefficient and has units of (ug/ml)-1cm1.
Molar Extinction
Coefficient
The values of E used here are as follows:
• ssDNA, 0.027 (ug/ml)-1cm-1
• dsDNA, 0.020 (ug/ml)-1cm-1
• ssRNA, 0.025 (ug/ml)-1cm-1
A Convenient Number to
Remember
• Using these calculations, an A260
of 1.0 indicates:
• 50 ug/ml double-stranded DNA
• ~ 37 ug/ml single-stranded DNA
• ~ 40 ug/ml single- stranded RNA
UV Quantitation of DNA
• The detection limit of absorption
spectroscopy will depend on the
sensitivity of the spectrophotometer
and any UV-absorbing contaminants
that might be present.
• The lower limit is generally ~0.5 to 1
ug nucleic acid.
Practical Use
• Measure some DS DNA in the Spec.
• Absorbance is too high.
• Dilute the DNA to get a reading in
range. Keep track of the dilution
factor (Example: 100 ul diluted in
900 ul is a 1/10 dilution with a
dilution factor of 10).
• Multiply 50 X 10 X OD=
concentration in ug/ml.
Practical Use
• Dilute some dsDNA 1/100 (dilution
factor of 100).
• Absorbance at 260 nm is 0.400.
100 X 0.4 X 50 = 2000 ug/ml
• What if it was RNA?
100 X 0.4 X 40 = 1600 ug/ml
Practical Use
• If you have 100 ul of DNA of
concentration at 2000 ug/ml, how
much RNA do you have?
Protein Contamination
• Proteins in general have A280
readings considerably lower than
nucleic acids on an equivalent
weight basis.
• Thus, even a small increase in the
A280 relative to A260 (Or a lowering of
the A260/A280 ratio) can indicate
severe protein contamination.
Spectral Properties of
Amino Acids
• Trp, Tyr, and Phe contain
conjugated aromatic rings.
• Consequently, they absorb light in
the ultraviolet range (UV).
• The extinction coefficients (or molar
absorption coefficients) of these
three amino acids are:
Amino
acid
Extinction Coefficient
Trp
5,050 M-1cm-1 (280 nm)
 (lmax)
Tryptophan
Tyr
1,440 M-1cm-1 (274 nm)
Tyrosine
Phe
Phenylalanine
220 M-1cm-1 (257 nm)
Proteins and
Spectrophotometer
• Trp absorbs UV light the strongest.
• Furthermore, since both Trp and Tyr
show the maximum light absorbance
at approximately 280 nm the
absorption maximum of most
proteins is around 280 nm.
• In contrast, the absorption maximum
for nucleic acids is approximately
260 nm.
Properties of Absorbance and Fluorescence
Spectrophotometric Assays for DNA and RNA
Absorbance
Property
Fluorescence
(A 260)
H33258
EtBr
DNA
1-50
0.01-15
0.1-10
RNA
1-40
n.a.
0.2-10
Ratio DNA/RNA
0.8
400
2.2
Sensitivity
(ug/ml)
Fluorescent Quantitation
of DNA
• For the Hoechst 33258 and ethidium
bromide assays, a plot of relative
fluorescence units or estimated
concentration (y axis) versus actual
concentration (x axis) typically produces a
linear regression with a correlation
coefficient (r2) of 0.98 to 0.99.
• Be certain that the final amount of DNA
does not exceed the linear portion of the
assay.