[6] Q U A N T I F Y I N G NUCLEIC ACIDS 49

[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

Recombinant DNA technology depends on detection methods for nu-

cleic acids compatible with amounts ranging from picograms to grams and
from tenths of a microliter to liters. In practical terms there are three basic
techniques: (1) absorbance methods suitable for a minimum concentration
of micrograms per milliliter, (2) fluorescence methods capable of detect-
ing nanograms of DNA and micrograms of RNA, and (3) methods based
on the detection of 32p. Because of the overwhelming importance in mo-
lecular biology of the third group, this chapter will stress exquisitely
sensitive methods for measuring radioactivity in very small volumes. ~,2
An illustration in which an enzyme-catalyzed reaction performed in 20/xl
is monitored by consuming less than 2% of the total volume will be pre-
sented.

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

I S. L . B e r g e r , Anal. Biochem. 136, 515 (1984).

2 S. L . B e r g e r a n d M. S. K r u g , BioTechniques 3, 38 (1985).
3 I. T i n o c o , Jr., J. Am. Chem. Soc. 82, 4785 (1960).

Copyright © 1987 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 152 All rights of reproduction in any form reserved.
50 ISOLATING AND CHARACTERIZING NUCLEIC ACIDS [6]

with a 1-cm path length is indicative of double-stranded DNA at a concen-

tration of approximately 50/xg/ml or of RNA or single-stranded DNA at
approximately 40/xg/ml. The absorbance of oligonucleotides is usually
the sum of the absorbance of the constituent mononucleotides (see this
volume [47]). A mixture of random oligomers at 20/.~g/ml has an absor-
bance at 260 nm of I.
The ratio of the absorbance at 260 to 280 nm is a useful indication of
purity. Values for DNA solutions of 1.8 to 1.9 and for RNA solutions of
1.9 to 2.0 are acceptable. The presence of protein, which absorbs at 280
nm, decreases the ratio as does phenol, another likely contaminant.

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.

Other Methods for Measuring Radioactivity

Recently an inexpensive method for counting 32p dried on filters has
become available. The device, Model QC 2000 manufactured by Bioscan
(Washington, DC), is a calibrated Geiger tube with a sample holder that
maintains a fixed geometry between the filter and the window of the tube.
The apparatus responds to/3 particles entering the window and measures
32p with a counting efficiency of - 3 0 % . It is about 10% the price of a
scintillation counter and takes up about a square foot of bench space.

4 j. V. Jelley, "Cerenkov Radiation and its Applications." Pergamon, Oxford, 1958.

[6] QUANTIFYING NUCLEIC ACIDS 53

Note that the instrument measures charged particles and n o t Cerenkov

radiation.

Procedure for Monitoring Reactions

The analysis of reactions with submicroliter quantities requires that
three conditions be met. One must be able (1) to determine the amount of
radioactivity on dry filters; (2) to process the dried samples, that is, wash
with TCA or phosphate to remove unincorporated labeled substrates; and
(3) to count the identical filters again. The procedure is as follows.
1. Assemble the components of the reaction in an Eppendorf tube
placed in an ice bucket.
2. With a Pipetman (Rainin) set at 0.2/xl, withdraw the smallest possi-
ble aliquot and spot it on the appropriate filter. (Use GF/C glass fiber
filters for macromolecules or DE-81 filters for oligonucleotides.)
3. Incubate at the prescribed temperature. As the reaction proceeds,
or at the anticipated time of completion, withdraw and spot additional
aliquots on separate filters.
4. Dry all filters under a heat lamp for 2 min. Measure the radioactiv-
ity on the dry filters using the 3H channel of a scintillation counter or the
Bioscan counter.
5. Wash the filters with 10 ml of solution. (Use 5% TCA containing 20
mM sodium pyrophosphate for glass fiber filters or 0.5 M sodium phos-
phate at pH 7 for DE-81 filters.) Wash GF/C filters with 1 ml ethanol to aid
in drying.
6. Dry the filters under heat lamps for 5 min and recount them using
either Cerenkov radiation (3H channel), the Bioscan counter, or scintilla-
tion fluid (rip channel). The last has a counting efficiency of almost 100%
and should be used for samples with low count rates.
7. Calculate the results.
An example taken from a nick translation reaction performed in a total
volume of 20 p.1 is shown below. To exhibit the versatility of the method,
the calculation will be carried out without referring to the amount of
radioactive substrate initially added. Assume it is unknown.
The data are as follows. The zero time filter, unwashed and washed,
contained 596,000 and 6,670 cpm, respectively; the 60-min filter, un-
washed and washed, contained 490,000 and 330,000 cpm, respectively.
All measurements were performed with dry filters at a counting efficiency
of 25%. By inspection of the filter obtained after 60 min of incubation, it
can be seen that 68% of the precursor was incorporated into DNA (the
background at zero time was negligible).
54 ISOLATING AND CHARACTERIZING NUCLEIC ACIDS [6]

Since the total radioactivity in the mixture is unknown, the radioactiv-

ity measurements obtained from the unwashed filters cannot immediately
be converted to volumes. In order to determine the amount of radioactive
substrate included in the reaction, the Cerenkov radiation that arises in
solution will be utilized. Toward this end, after completing the nick trans-
lation, the original 20-/xl volume was diluted with 130/~l of buffer to make
a final volume of 150/zl. (The amount withdrawn during the incubation
was trivial and was ignored.)
8. To each of three 1.5-ml Eppendorf tubes, add 1/zl of the complete
diluted reaction mix. Centrifuge briefly to drive the drop to the bottom.
9. Suspend each Eppendorf tube on the rim o f a 20-ml scintillation vial
and count in the 3H channel. Since the Cerenkov radiation arises in water
with improved geometry, rather than in glass, the counting efficiency is
now 56%. 5 Note that it is necessary to dilute the reaction so as not to
exceed the linear range of a scintillation counter.
10. Recover the three aliquots and return them to the original tube.
They are not consumed.
From the average radioactivity of the liquid samples, namely, 1.07 x
106 cpm//zl at 56% efficiency5 (which is 4.75 × IVs cpm//xl at 25% effi-
ciencyS), the total radioactivity in the nick translation reaction can be
determined. Thus, 4.75 x 105 cpm//xl × 150/~1 or 7.12 x 10 7 cpm (25%
efficiency) was initially added. This value is equivalent to 130/xCi of
substrate. It follows that the volumes applied to filters were 0.167 /zl
(5.96 x 105 cpm x 20 ~1/7.12 x l o s cpm) and 0.138/xl (4.90 x 105 cpm x
20/xl/7.12 x 10 7 cpm), respectively, at 0 and 60 min of incubation. The
reaction generated 3zp-labeled DNA containing 1.90 x I08 dpm, correct-
ing for the zero time value. The complete analysis of the reaction con-
sumed 0.305/xl or 1.5% of the total sample. Clearly, small reaction vol-
umes should n e v e r deter the investigator from analyzing any reaction in
which 32p-labeled substrates can be separated from 32P-labeled products
by a filter assay.

s T h e efficiency o f m e a s u r i n g C e r e n k o v radiation on dry filters or in solution m u s t be

d e t e r m i n e d for each i n s t r u m e n t .