Nucleic Acids - Volume 002 of Methods in Molecular Biology - Humana Press

367 Pages·2016·16.11 MB·English

by  Walker J.M.

Chapter 1
The Burton Assay for DNA
Jaap H. Waterborg and
Harry R. Matthew
Department of Biological Chemisty, University of
California School of Medicine, Davis,
California
Introduction
The Burton assay for DNA is a colonmetric procedure
for measuring the deoxyribose moiety of DNA. It is rea-
sonably specific for deoxyribose, although very high con-
centrations of ribose (from RNA) or sucrose must be
avoided. The method can be used on relatively crude ex-
tracts and m other circumstances where direct measure-
ment of ultraviolet absorbance of denatured DNA is not
practical. The assay has been widely used.
Materials
1. Diphenylamine reagent: Dissolve 1 5 g of diphenyla-
mme m 100 mL glacial acetic acid. Add 1 5 mL of con-
centrated (98--100%) H2S04 and mix well. Store this re-
agent m the dark. Just before use, add 0.5 mL of
acetaldehyde stock solution.
1
2 Waterborg and Matthews
2 Acetaldehyde stock. 2 mL acetaldehyde m 100 mL drs-
tilled water. Store at 4°C where rt IS stable for a few
months.
3 1N perchlonc acid (PCA).
4. Standards. Dilute a DNA stock solutron with drstrlled
water as follows.
DNA stock, /.I,L 0 10 20 50 100 200
Water, mL 10 0 990 0 980 0 950 0.900 0 800
DNA concen-
tration, j.@mL 0 10 20 50 100 200
5 DNA stock 1 mg/mL u-r distrlled water. Store frozen at
-20°C where rt IS stable for a few months
Method
1. Extract the sample as required (see Notes)
2. Add 0.5 mL of 1N PCA to 0 5 mL of sample or
standard. Hydrolyze for 70 mm at 70°C.
3. Cool the hydrolyzed samples on ice for 5 mm. Centri-
fuge (15OOg; 5 min; 4°C) and decant the supernatants
mto marked tubes.
4. Add 1 mL of 0.5N PCA to each pellet, vortex, repeat
step 3, and carry the combmed supernatants forward to
step 5. (This step IS optronal, see Note 3)
5. Add 2 vol of drphenylamme reagent to 1 vol of the su-
pernatants (0 5N PCA hydrolyzates from step 3). MIX
and incubate at 30°C for 18 hr.
6. Read the absorbance at both 595 and 650 nm, using the
0 bg/mL standard as a blank
7 Plot a standard curve of absorbance at 595 nm mmus
absorbance at 650 nm as a functron of mrtral DNA con-
centration and then use the curve to read off unknown
DNA concentratrons.
Notes
1. Extraction condrtrons may have to be optrmrzed for par-
ticular applications. The followmg procedures are
routinely used m our laboratory
Burton DNA Assay 3
(a) This extraction is required if the sample contams
mercaptoethanol, dithiothreitol, or other inter-
fering low molecular weight substances. It is also
required as a preliminary if extraction (b), which is
for whole cells or organelles that contam lipids, is
to be used. Add 1 vol. of 0.2N PCA m 50% etha-
nol : 50% distilled water and mix by vortexing Cool
on ice for 15 min and then centrifuge (5 mm, 15OOg,
4°C). Discard the supernatant.
(b) To the pellet add 1 mL of ethanol-ether (3: 1, v/v).
Incubate for 10 mm at 70°C Centrifuge (5 mm,
15OOg) and discard the supernatant. To the pellet
add 1 mL of ethanol (96%), vortex and centrifuge
(5 mm, 15008). Discard the supernatant.
2. If the sample is a pellet, at step 2 add 1 mL of 0.5N PCA
to the pellet and proceed with the hydrolysis at 70°C. If
the sample is too dilute ( < 10 kg/mL) and is available
m a volume larger than 0.5 mL, then it may be concen-
trated by precipitation, as described m Note 1, extrac-
tion (a), or by precipitation with 0.5N PCA
3. Step 4 is optional It provides a more quantitative re-
covery of nucleic acid m the supernatant, but reduces
the sensitivity of the overall assay.
4. The diphenylamme reagent is not water soluble. Rinse
out glassware with ethanol before washing In water.
Take care to use a dry spectrophotometer cuvet and
clean it with ethanol.
5. It IS recommended to run a standard curve with each
group of assays, preferably in duplicate. Duplicate or
triplicate unknowns are recommended.
References
2 Burton, K (1956) A study of the condltlons and mechamsm
of the dlphenylamme reaction for the colorlmetrlc estlma-
tlon of DNA Blochem J 62, 315-323

Chapter 2
DABA Fluorescence Assay
for Submicrogram
Amounts of DNA
Theodore Gurney, Jr. and
Elizabeth G. Gurney
Department of Biology, Unwersdy of Utah, Salt Luke
City, Utah
Introduction
The fluorescence assay of Krssane and Robins (1) 1s
used to quantify deoxypurme nucleosldes m crude
mixtures. Acid-catalyzed depurmation exposes the 1’ and
2’ carbons of deoxyribose, which can then form a strongly
fluorescent compound with diammobenzolc acid (DABA)
DABA can react with all aldehydes of the form
RCH,CHO, but deoxyrrbose is the predominant one m
mammalian cells and essentially the only one m the acid or
alcohol precipitates of aqueous extracts Hence, no purrfr-
cation is required and RNA does not interfere. In our
hands, the method IS useful down to 30 ng of DNA, and
probably could be made more sensmve, as discussed be-
low. The method requires a visible-light fluorometer, the
excltatlon wavelength IS near 410 nm, with maximum fluo-
rescence near 510 nm (2)
5
6 Gurney and Gurney
Materials
1. The purity of DABA (3,5-drammobenzorc acid
drhydrochlorrde) determines the background of the
assay, and hence the sensitivity Purified DABA, m
drhydrochlorrde form, can be purchased, or else crude
commercral material can be purified. DABA should be
white with a slight yellow-green fluorescence. Brown
or grey powder usually gives high background. A
procedure for purifying DABA 1s given below. DABA
IS stored in powder form at -20°C and IS stable for
years.
2. Highly purified mammalian DNA or salmon sperm
DNA 1s used to calibrate the assay DNA IS dissolved
m water at concentrations of 50, 100, and 300 kg/mL
and 1s stored m quantities of 1 mL at -20°C. One
set of the dilutions suffices for at least 20 cahbra-
tron curves. Concentrations of DNA are measured m
O.lM NaCl assuming Ey& = 200, 1 e , 10 kg gives
A 260 = 0 2.
3. Purified RNA IS used to coprecipitate DNA from di-
lute solutrons. Commerrcal yeast RNA, free from de-
tectable DNA, can be stored in powder form at -20°C.
RNA is used in a precipitation buffer, described
below.
4 A fluorometer or a spectrofluorometer. The excrtatron
wavelength 1s 410 nm and the emrssron wavelength IS
510 nm. Hmegardner (2) describes specific equrp-
ment.
5. Precipitatron buffer 100 mM NaCl, 10 mM Trrs HCl,
pH 7 5, 1 mM EDTA; 100 kg/mL yeast RNA The
buffer 1s stored at +2”C, or at -20°C rf rt is not used
frequently. Bacterial growths can certainly raise the
background
6. Trrchloroacetic acid IS prepared as a 20% (w/v) solu-
tion and stored at +2”C.
Methods
Purification of DABA
This procedure, based on that of Hmegardner (2), will
yield better DABA than the best commercral material,
DABA Fluorescence Assay for DNA 7
starting with mexpensive crude DABA. Yields should be
50-70% *
1. Put 100 g of crude (dark brown) DABA in a 1 L beaker,
add 250 mL of distilled water, and stir to dissolve at
room temperature m a fume hood.
2 Add 250 mL of concentrated I-ICI and stir slowly with
a glass rod A precipitate will form. Collect the precip-
itate on Whatman #1 paper using a Buchner funnel
and suction. The suspension is thixotropic, so it is
necessary to shake the funnel while filtering.
3 Redissolve the precipitate m 250 mL of water, or more
if necessary, and then add an equal volume (to that of
added water) of concentrated HCI
4. Filter as above. If necessary, repeat the sol-
ution-precipitation cycles untrl the color of the precip-
itate is no darker than light brown
5. Dissolve the precipitate m the minimum amount of
water necessary Measure the water volume, and then
add 15 mg of activated charcoal powder “Norit A” per
mL of added water. Stir to make a uniform suspen-
sion and then let the suspension rest unstirred for 30
mm.
6. Centrifuge the suspension (5OOOg,1 5 min, 2O”C), and
decant the supernatant. Do not worry if a little of the
charcoal is decanted. Discard the pellet.
7. Remove any residual charcoal by filtering through a
0 45 pm nitrocellulose filter with a cellulose prefilter
You might have to change filters because of blocking.
The filtrate should be clear.
8 Add an equal volume of concentrated HCl to the fil-
trate and stir gently. White crystals should form m a
light yellow fluid
9. Collect the precipitate on Whatman #1 and transfer to
a baking dish previously cleaned with HCI Chop up
the precipitate mto small pieces with a clean glass rod.
All surfaces touching DABA must be very clean from
this point.
10 Heat the open baking dish at 60°C m a fume hood
overnight, in the dark. This may be done by covering
a thermostatted waterbath on all sides with
polyethylene sheet before adding the dish. This pre-
vents evaporation of the water and protects the metal
Gurney and Gurney
parts of the waterbath from HCl vapor The dish must
be uncovered to allow HCl evaporation The DABA is
ready when there IS no more HCl odor.
11. Store powdered DABA tightly sealed m a brown lar at
-20°C. Allow the jar to warm to room temperature
before openmg.
Sample Preparation: Tube
Method
Two methods of sample preparation are given. The
tube method is preferred if the sample is available, salt-
free, m a volume of 100 PL or less. The sample may be
crude and does not have to be m solution, e.g., a suspen-
sion of whole cells. If the sample is too dilute, too salty, or
if you wish to remove soluble DABA-positive material,
then you should precipitate the sample first by using the
filter method described below (Note 1). The tube method
gives lower background
1. Spot the sample m the bottom of a 12 x 77 mm
polypropylene tube and dry it at 50°C. The dried
samples are stable for several days at room tempera-
ture, so you may accumulate several samples to assay
later.
2. Prepare six DNA standards, mcludmg a zero DNA
standard, m the same way as step 1, from the DNA so-
lutions Also prepare a blank sample with no DNA, but
with the manipulations and buffers you use in your ex-
perimental samples (Note 2) The amounts of DNA m
standards should bracket your experimental values.
Sample Preparation: Filter
Method
1. Spot the sample m a 12 x 77 mm polypropylene tube
and add 0.2 or 0.5 mL of precipitation buffer The final
nucleic acid concentration should be at least 50 pg/mL,
mostly yeast RNA from the precipitation buffer
2 Mix, then add TCA to 5 or 10% (w/v) final concentra-
tion, mix again, and chill on ice for 30 mm.
DABA Fluorescence Assay for DNA 9
0
/
a
:/
l
/
2-0 - 20 TUBE ASSAY i FILTER ASSAY
IL i
L
.I-..L-. I I I
05 10 2 4 6
/cg DNA
Fig 1 Salmon sperm DNA was prepared and assayed by the
tube method and the filter method
3 Filter the sample onto a GFK filter, rinsing the tube
and apparatus three trmes with 2 mL of cold 1N HCl.
Remove the chimney from the apparatus and rinse the
filter once with about 1 mL of 95% ethanol.
4. Remove the filter, wet with ethanol, to a flat-bottomed
glass vial and dry it there with the top off. The dry
samples are stable at room temperature for several
days.
5. Prepare DNA standards m the same way, on GF/C fil-
ters. Standards prepared by the tube method have a
lower background than those prepared by the filter
method (See Fig. 1)
The DABA Assay
1. Take the powdered DABA out of the freezer and let it
warm to room temperature. For each tube sample, you
will need 32 mg of DABA powder plus 80 FL of HZ0
For each filter sample, you will need 80 mg of DABA
powder plus 200 PL of HZ0 (Note 3)
2. Weigh the DABA, dissolve it m the appropriate volume
of water, and add 0.1 mL of this solution to each tube
or 0 25 mL to each filter.
3 Incubate the samples at 55-57°C for 45 mm (Note 4),
then dilute the samples with l-3 mL of 1iV HCl (Note
5).