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6 Quantifying 32P Labeled and Unlabeled Nucleic A - 1987 - Methods in Enzymolo
6 Quantifying 32P Labeled and Unlabeled Nucleic A - 1987 - Methods in Enzymolo
6 Quantifying 32P Labeled and Unlabeled Nucleic A - 1987 - Methods in Enzymolo
[6] Q u a n t i f y i n g 3 2 p - L a b e l e d a n d U n l a b e l e d N u c l e i c A c i d s
By SHELBY L. BERGER
Absorbance Methods
Both DNA and RNA absorb in the ultraviolet between 250 and 270 nm
owing to the spectral characteristics of the four canonical bases. Near
neutrality, the molar extinction coefficients of the bases and the wave-
length at which maximum absorption occurs are as follows for a 1-cm path
length: 1.54 × 10 4 (259 nm) adenine, 9.1 x 103 (271 nm) cytosine, 1.37 x
104 (253 nm) guanine, 1.0 x 104 (262 nm) uracil, and 7.4 x 103 (260 nm)
thymine. (Molar extinction coefficients of the four bases at 260 nm can be
found in this volume [47].) These values are useful for determining the
concentrations of solutions of nucleoside triphosphates. When the ab-
sorption spectrum of either DNA or RNA is compared with the sum of the
absorption spectra of the constituent purines and pyrimidines, however,
there is a discrepancy: in typical DNA preparations, the intensity of ab-
sorption may be 40% less than the intensity of a mixture of the corre-
sponding nucleotides at the same wavelength. This "hypochromic effect"
is caused by the interaction between absorbing units when placed in an
orderly array. 3 Thus, at 260 nm, an absorbance of 1 measured in a cuvette
Fluorescence Methods
DNA and R N A are not themselves fluorescent. When ethidium bro-
mide binds to nucleic acids in solution, the dye, upon excitation in the
ultraviolet, fluoresces intensely in the visible range. The orange fluores-
cence can be used for quantifying both double-stranded and single-
stranded molecules, but with markedly different sensitivity. Usually, this
goal is achieved by subjecting the sample DNA, together with a series of
marker DNAs at different concentrations, to electrophoresis in a gel. The
gel is stained with ethidium bromide at 1/~g/ml for 30 min and viewed with
a UV transilluminator. (See this volume [8] for a more detailed discus-
sion.) Since bands of equal intensity contain the same mass of material
regardless of the size of the DNA, the amount of sample DNA can be
estimated by visual comparison with the known standards. Although the
eye is adept at matching near-equal intensities, it cannot estimate inten-
sity differences well nor compare a diffuse signal with a sharp one. There-
fore, the most precise comparisons are between markers and samples that
have size and intensity in common. This approach is capable of detecting
- 5 ng of double-stranded DNA and - 0 . 5 / x g of RNA depending on the
width and thickness of the band. (See this volume [8] for a more detailed
discussion.) Quantitative evaluation of RNA requires marker RNAs or
single-stranded DNAs at known concentrations. Methods for staining de-
naturing RNA gels can be found in this volume [8].
Solution methods for measuring fluorescence have not enjoyed wide
popularity. Although potentially quantitative, the signal obtained from
ethidium bromide in a solution of DNA depends on the degree of super-
coiling of the DNA and the degree of quenching caused by inadvertant
contamination. Because neither of these can be assessed easily, most
investigators continue to rely on the semiquantitative methods described
above. (See this volume [55] for a description of the technique.)
[6] QUANTIFYING NUCLEIC ACIDS 51
32p-Based Methods
Many of the reactions used to clone and characterize nucleic acids
must be performed in small volumes in order to obtain adequate concen-
trations of enzymes and substrates. Frequently, these reactions involve
the incorporation of 32P-labeled substrates into 32p-labeled DNA or RNA.
Such reactions can be fully monitored by withdrawing submicroliter quan-
tities for analysis; the majority is retained for further manipulation.
The technique which follows makes use of a filter assay to separate
32p-labeled precursors, such as nucleoside triphosphates or pCp, from
labeled polynucleotides. It is based on the observation that precipitation
with trichloroacetic acid (TCA) need not be performed in solution. It can
be performed equally well by placing a minute drop (-0.1 to 0.2 ~1) of
material consisting of a mixture of labeled substrates and products on a
glass fiber filter, drying the filter, and washing it with TCA. The acid-
soluble substrates are eluted leaving the labeled macromolecules on the
filter for subsequent assay in a scintillation counter. (Filtration equipment
is described in this volume [1].)
For oligonucleotides too small to be acid precipitable, an analogous
technique can be used by substituting DE-81 filters for the glass fiber
variety and 0.5 M phosphate at pH 7 for TCA. Oligonucleotides adhere to
the filter while nucleotides are removed by washing with the buffer.
The ability to evaluate the progress of a reaction by withdrawing sub-
microliter volumes depends on methods for assaying radioactivity on dry
filters. Cerenkov radiation, which occurs within the glass or plastic wall of
a scintillation vial when a dry filter--no liquid whatsoever--is placed flat
on the bottom of the vial, is a measure of radioactivity. By determining
Cerenkov radiation with the 3H channel of a scintillation counter, one can
assay 3zp with approximately 25% counting efficiency. The existence of
Cerenkov radiation, then, makes possible measurements of radioactivity
on filters in a nondestructive manner before they are processed; the
counting process has no effect on subsequent procedures performed with
the same filter.
It should be clear that the amount of radioactivity spotted on a filter
and dried is proportional to the volume applied to that filter. The propor-
tion is valid at any time during a reaction, since it is independent of
whether substrates have been converted to products. In contrast, the
radioactivity on filters washed with TCA or phosphate buffer reflects only
the products of the reaction. With two or more filters, one obtained at
zero time and the others after incubation for predetermined intervals, the
reaction can be characterized. Given the amount of radioactive substrate
52 ISOLATING AND CHARACTERIZING NUCLEIC ACIDS [6]
initially included in the incubation mixture, the total volume in which the
reaction is being conducted, and the efficiency of counting dry filters, one
can calculate the precise volume spotted from the radioactivity on each
unwashed filter. Given the radioactivity remaining after processing the
identical filters, the amount of product per unit volume can be ascer-
tained. The ratio of radioactivity on a washed filter to that of the same
filter before it was washed is a measure of the percentage incorporation of
precursor into product. Since the precise volume spotted is determined
from radioactivity measurements and not from the designated volume of a
micropipet, the smallest possible aliquot should be withdrawn, knowing
that subsequent withdrawals will result in different but nevertheless pre-
cisely measurable volumes.
Cerenkov Radiation
Although a detailed description of Cerenkov radiation is beyond the
scope of this chapter, 2,4 it may be useful to summarize the salient features.
Cerenkov radiation occurs when a charged particle in a dielectric medium
travels faster than the phase velocity of light in that medium. (It is under-
stood that the velocity of the particle cannot exceed the velocity of light in
vacuo.) Then, a series of pulses of light are emitted that are analogous to
the V-shaped pattern of sonic booms produced by aircraft traveling at
supersonic speeds. These optical " b o o m s " occur in the visible or near-
visible regions of the spectrum with a fixed geometry relative to the mov-
ing particle. Like the sonic boom, there is a threshold velocity of the
particle below which Cerenkov radiation cannot take place. Thus, ener-
getic 32p particles give rise to Cerenkov radiation in the wall of a scintilla-
tion vial or in solution, each at a different efficiency. Both modes can be
used to advantage.