Download Separation of DNA Restriction Fragments by Ion

Separation of DNA Restriction Fragments by Ion-Exchange
Chromatography
on FPLC Columns Mono P and Mono Q
ELISABETH
WESTMAN,
STIG ERIKSSON, TORGNY L,Us,’
AND SVEN-ERIK SKOLD
Received
March
PER-AKE
PERNEMALM,
9, 1987
Separation
of DNA restriction
fragments
by FPLC ion-exchange
chromatography
on Mono
Q and Mono P columns was investigated.
The columns
were found to be particularly
suitable
for the separation
of fragments
up to 500-600
bp long. Larger fragments
can also be separated
although
less effectively.
We found the following
practical working
ranges for the parameters
investigated:
pH, 4 to 1 I; flow rate. 0.05 to 0.6 ml/min
corresponding
to separation
times
between 2 and 20 h. (better resolution
is achieved at lower flow rates); gradient slope: between
0.5 mM eluting salt/ml buffer and over 5 mM/ml
(better resolution
is achieved at lower gradient
slopes; eluting ionic strength was found to be independent of gradient slope): gradient composition. chloride salts of smaller monovalent
cations eluted the DNA at lower ionic strengths but
separations
obtained
were similar: additives,
substances such as urea, formamide,
and EDTA
can be added without
chromatographic
effects; sample amount:
amounts
from 2.5 to 200 Gg
were applied. corresponding
to single peak content of from 42 ng to 74 pg DNA. Yields were
generally over 90% and the chromatographed
DNA was fully accessible to restriction enzyme
cleavage. Separations
occurred
predominantly
according
to DNA size. but AT-rich
fragments
were retarded in a predictable
way.
c 1987 Acadrmic press. IIK.
KEY WORDS: chromatography.
nucleic acids; FPLC. nucleic acids; DNA; Nucleic acid chemistry; HPLC, techniques.
The standard method today for the separation of DNA restriction fragments is electrophoresis in polyacrylamide and agarosegels.
Considering that electrophoresis has unmatched separation power, is relatively easy
to perform with inexpensive equipment, and
is suitable for parallel separations of many
samples, it is natural that electrophoresis
plays a central role in DNA research. However, when used for preparative purposes
electrophoresis has two particularly serious
drawbacks:
(A) Extraction of the purified material out
of the gel is tedious and difficult to accomplish with high yields (1).
(B) The purified DNA is often contaminated with agarose impurities having enzyme-inhibiting properties (2-5). This prob’ To whom
0003-2697/87
correspondence
should
$3.00
Copyright LB lY87 by Academic Press, Inc.
All rights ofrcproduction
in any form reserved.
be addressed.
158
lem is now reduced with commercially available “nucleic acid-grade” agarose.
Chromatographic procedures for preparative DNA separations would circumvent
these drawbacks and several attempts have
also been made to separate fragments on different chromatographic media. Chromatographic methods that have been investigated
for the separation of DNA fragments include
gel filtration (6-8), mixed-mode separations
(combined reversed phase and ion exchange)
(9), reversed phase (RPC) ( lo), ion-paired
reversed phase (I 1). 2-phase partitioning
( 12,13), and ion-exchange chromatography
(14-18).
In this work we have studied the separation of restriction fragments on the commercially available anion-exchange supports
Mono Q and Mono P. There are several reasons for the choice of these columns:
ION-EXCHANGE
SEPARATION
(A) They have a documented
ing power for proteins ( 19).
high resolv-
(B) They have a high potential for nucleic
acid separation as indicated by our preliminary investigation (20).
(C) They have suitable chemical
and
physical properties (19). As opposed to the
silica-based supports, Mono P and Mono Q
are not alkali labile. Furthermore,
the ion-exchange groups are covalently linked to the
supports and cannot contaminate the sample
as, for example, the adogene (methyltrialkyl
(C8-c 10) ammonium
chloride)
from
RPC-5-type
materials.
Another
attractive
feature is their macroporosity.
(D) The fact that the two ion-exchange
materials are made from the same basic support yet have differences should make possible comparisons between the relative importance ofthe basic support versus the charged
substituents:
Mono Q is a strong ion exchanger with a high density of quaternary
amino groups (0.27-0.37 mmol/ml).
Mono
P, which was designed for chromatofocusing
(19), contains
a range of different
weak
amino groups with pK values evenly spread
from ca. pH .3 up to over pH 9. The charge
content of this gel will thus change with pH.
At low pH the gel will be highly charged,
while at high pH it will hardly be charged
at all.
In particular we wanted to compare the
effects of the following factors on the separations on the two columns: (i) buffer composition and pII. (ii) flow rate, (iii) gradient
composition and slope. (iv) presence of additives. We were also interested in the amounts
of sample that can be processed and the efficiency of the separations
in terms of the
yield, purity. and quality of the chromatographed DNA.
MATERIALS
AND
METHODS
C’hemicafs. All DNA samples and enzymes were sulpplied by Pharmacia. All other
chemicals were of analytical grade.
OF
RESTRICTION
FRAGMENTS
159
C’hromatogruph~~. The Pharmacia FPLC
system (pump P500, liquid chromatography
controller LCC 500, uv monitor UV- 1, fraction collector Frac 100. recorder REC-482,
and Mono P and Mono Q columns) was set
up and used for gradient elution, fraction
collection, and peak evaluation essentially
according to the manufacturer’s instructions.
E1~~~tro~phor.csi.c.
Agarose gel electrophoresesaccording to standard protocols were performed on the Pharmacia GNA 100 apparatus in agarose NA (nucleic acid-quality
agarose, Pharmacia). Restriction fragment
patterns were also analyzed by polyacrylamide gradient gel electrophoresis on precast
gradient gels (Pharmacia gradient gels PAA
2/16) in the Pharmacia GE 2/4 vertical electrophoresis apparatus.
Visualization of DNA bands on both agaroseand acrylamide gelswas accomplished bq
fluorescence on a uv table (ultraviolet products Ltd.) after soaking the gels in ethidium
bromide solutions (0.5-I @g/ml) for ca.
0.5-l h.
Etz:~~ne rwcfiom. The standard sample
was obtained by digesting plasmid pBR322
with Hi&I. This results in fragments of 163 1,
5 17, 506. 396, 344. 298, 221, 220, 154, and
75 base pairs.
Another mixture of fragments between 7
and 587 base pairs (7, 11, 18. 21, 5 1, 57. 64,
80, 89, 104, 123, 124, 184. 192, 213, 234,
267,458.504.540. and 587 bp) was obtained
by treating pBR332 with I~~eIII.
The quality ofthe chromatographed DNA
was checked by observing its accessability to
cleavage with I:‘coRI.
All enzyme reactions were performed according to the manufacturer’s instructions.
Qlluntificution.
The amount of eluted
DNA was estimated by automated integration by the FPLC equipment as well as by
fluorescent spectroscopy using an Aminco
SPF-500 fluorescence spectrophotometer essentially as previously described (3 l-23): To
a l-ml sample in 10 mM Tris-HCI, 1 mM
EDTA. pH 8.0. was added 150 11 propidium
iodide ( 100 pg/ml in the same buffer). The
160
WESTMAN
FIG. 1. Separation
of-?0 big ifjnfl-digested
were 20 mM piperazine-HCI,
1 rnM EDTA,
respectively.
Elution was accomplished
by
buffer A (pure buffer) with buffer B (buffer
ET AL.
pBR372 on Mono Q at pH 6.0 (a) and pH 9.0 (b). Buffers used
pH 6.0, and 10 mM ethanolamine-HCI,
I mM EDTA, pH 9.0,
a linear KC1 gradient (2.8 mM/ml
buffer) obtained by mixing
containing
I M KU). The flow rate was 0. I5 ml/min.
fluorescence at 620 nm was measured after
excitation at 550 nm. The amount of DNA
was estimated by comparison with the fluorescence from known amounts of pBR322.
Identijcation.
The identity of the separated peaks was determined by comparing
the positions after agarose electrophoresis
with the positions of fragments of known
size.
RESULTS
The primary aim of this work was to compare the effect of different experimental conditions on the separation of DNA fragments
on Mono Q and Mono P columns. For this
purpose we separated the same test sample
(Hinff-digested
pBR322) on the two columns under systematically
varied conditions.
Bufer pH
The separations obtained on Mono Q and
Mono P in the neutral pH range were compared. Typical results are shown in Figs. 1
and 2.
As can be seen. the separation profiles are
quite independent of pH in the neutral pH
range. Almost identical chromatographic
patterns were also obtained with 20 mM bisTris propane-HCI
(pH 7.0) and Tris-HCl
(pH 8.0). The only significant difference is
that the ionic strength needed for elution
from the Mono P column is strongly pH dependent. At lower pH, where the column has
a higher charge, a higher ionic strength is
needed. At a pH of 3.8 the DNA fragments
were eluted between ca. 1.1 and 1.2 M NaCl,
while at pH 10.0 elution took place between
ca. 0.5 and 0.6 M NaCl. This pH dependence
is more clearly shown in Fig. 3. where the
elution position of two selected fragments is
plotted for various pH values.
Standard buffer for most subsequent experiments was 20 mM Tris-HCl, pH 8.3 (for
Mono Q) or 8.0 (for Mono P).
Flow Rate
The standard sample was separated at different flow rates on the Mono Q and Mono P
columns. Typical results are illustrated in
Figs. 4 and 5.
Figures 4 and 5 illustrate typical separations at extreme flow rates. Generally it was
found that the resolution obtained at low
ION-EXCHANGE
SEPARATION
OF
RESTRICTION
FRAGMENTS
161
FIG. 2. Separation
of Ilk&I-digested
pBR323 (20 and 10 pg. respectively)
on Mono P at pH 6.0 (20 mM
histidine-HCl)
and pH 9.0 (20 mM ethanolamine-HCI)
(a and b respectively).
In (a) buffer A contained
0.9 M NaCl and buffer B contained
1. I M. In (b) buffer A contained
0.53 M NaCl and buffer B contained
0.70 M. The gradient slope was 2.8 mM/ml
and the flow rate was 0. I5 ml/min
in both cases.
flow rates deteriorated only slowly when the
flow rate was increased to moderately high
values. However. at flow rates of about 0.3
ml/min for Mono Q and ca. 0.6 ml/min for
Mono P dramatic changes in the chromatographic pattern occurred. The fragment
started to elute at lower ionic strength. an
FK. 3. The concentration
of NaCl needed for elution
of the 154- and 163 I-bp fragments
from Mono P as a
function
of the pH of the buffer.
Besides the buffers
already mentionesd
the following
buffers were used: 20
mM
sodium acetate. pH 3.9 and 5.0: 20 mM imidazoleHCI, pH 7.5: 20 mM piperazine-HCI,
pH 9.6 and 10.1:
and 20 mM sodium bicarbonate.
pH I I .2.
effect that was more pronounced the larger
the fragment size and the higher the flow
rate. This resulted in compressed chromatograms with decreased resolution at high flow
rates as illustrated by Figs. 4b and 5b.
In order to get an objective picture of the
dependence of resolution on flow rate, Fig. 6
was composed. This figure shows the resolution between two relatively small and two
relatively large fragments on both columns
as a function of flow rate.
Grudient Slope
Separations were performed on both columns with NaCl gradients ranging from 0.5
to more than 5 mM/ml. The NaCl concentration for elution of a particular fragment
was always the same regardless of the gradient slope. A typical steep gradient separation is demonstrated in Fig. 7.
Data from several separations at different
gradient slopes were collected and the resolution between the same two pairs of fragments
was calculated and plotted (Fig. 8).
Gradient Composition
To study the effect of the eluting salt on
the Mono Q column, we first varied the cat-
162
WESTMAN
ET
”
AL.
b
A254
0.09
NaCl
1.0
M
t
FIG. 4. Separation
of20 pg of the standard sample on Mono Q at low tlow rate. 0.05 ml/min
(a). and at
high flow rate. 0.5 ml/min
(b). The buffer used was 20 mM Tris-HCI.
I mM EDTA, pH 8.3. Elution was
accomplished
with a linear gradient of NaCl(2.8
mM/ml buffer), obtained by mixing buffer solutions with
0.5 and I .O M NaCl respectively
ionic component while keeping chloride as
the anion. The cations studied were Li+,
Na+, K+, and Cs+. All salts gave similar chromatographic separations. Possibly Cs+ gave
slightly better resolution of the larger fragments. The only significant difference between the different salts was that smaller ions
A254
eluted the DNA at lower ionic strengths than
did the larger ones. The results are summarized in Fig. 9. Attempts to use the sodium
salts of acetate and trichloroacetate or sulfate
as desorbing agents gave unsatisfactory results. With the trichloroacetate
very poor
separations were obtained and sodium sul-
4
A254
NaCl
0.04
a
I
.- l.OM
0.04
t
,631
.. 0.02
20
60
40
ml
)
1
2
3
FIG. 5. Separation
of 20 pg standard sample at low flow rate. 0.05 ml/min (a). and at high flow rate, 0.6
ml/min (b), on Mono P in 20 mM Tris-HCI,
1 mM EDTA, pH 8.0. Elution was accomplished
with a linear
NaCl gradient. 2 mM/ml
buffer. The gradient was obtained by mixing buffer solutions containing
0.72 and
0.86 M NaCl respectively.
h
ION-EXCHANGE
15.0
SEPARATION
MO”0
0 (
MO”0
P ’
0 Mono
. MOnO
75-154
0,
p, 506-517
bp
bp
10.0
OF
RESTRICTION
FRAGMENTS
163
In another experiment on Mono Q, the
amounts of DNA in all peaks were compared
with the total applied amount of DNA (37
pg) by fluorescence spectroscopy. The total
recovery was 93%. All fragments gave recoveries in the range of 90-95s with no significant difference between small and large fragments.
5.0
0
0
0.5
1.0
Flow
rate
(ml/mid
FIG. 6. Resolution
between the 75 and 154-bp fragments on one hand and the 506- and 5 17.bp fragments
on the other on Mono Q and Mono P, respectively,
as a
function of flow rate. Resolution
was calculated
according to the standard formula
R = 21/(sB, + M.& where f is
the distance between the two peaks and M’, and M’> is the
width of the two peaks. Data were collected from scparations at the flow rates indicated.
fate failed to elute the DNA, even at concentrations as high as 2. M.
As an illustration of the capacity of the
Mono Q and Mono P columns, Fig. 12
shows the separation of a 0.2-mg sample.
Figure 13, on the other hand, showsthe separation of a 2.5clg sample and demonstrates
the detection sensitivity of the system.
Both ligation of purified fragments and
cleavage with a number of restriction enzymes proceeded quite normally, as illustrated in Fig. 14 for EcoRI.
Among the most common additives in
nucleic acid work are urea, formamide.
EDTA. and Mg’+. Our experience with the
use of these aclditives in Mono Q and Mono
P chromatography is summarized below:
Urea: Even high concentrations of urea do
not seem to allfect the quality of the separations significantly. However, urea does seem
to modify the interaction between the DNA
and the chromatographic support since considerably shallower gradients must be used
(Fig. 10).
Formamide (up to 2 M), EDTA (I mM),
and Mg” (10 mM) had no effect on the separation.
Rechromatography
of individual fragments and comparison of the eluted
amounts generally gave recoveries well over
90%. A typical result from rechromatography on Mono I’ is illustrated in Fig. 11.
FIG. 7. Steep gradient
separation
on Mono Q. Seven
micrograms
of Hi&l-digested
pBR373 were chromatographed at a flow rate of 0. I5 ml/min.
The gradient was
created by mixing bufTer (containing
0.5 M NaCI) with
buffer (with I .O M NaCl) to a gradient slope of‘5 mM/ml
buffer.
WESTMAN
164
ET AL.
b54
0.019
Nacl
1.0M
t
1
--o.o1631
;I
-0.006
221
0.5
506 517
220
29P4
396
154
75
20
FIG. 8. Separation
between the 75 and 154-bp fragments and the 506- and 5 17-bp fragments,
respectively.
on the Mono Q and Mono P columns
as a function
of
gradient slope. Resolution
was calculated
as described in
the legend to Fig. 6. Experimental
conditions
were the
same as in Fig. 7 except for gradient
slope. which was
varied.
40
i
60
i
ml
3
i
h
FIG. 10. Separation
of 5 pg standard sample (Ilinfl-digested pBR322)
on Mono P in 20 mM Tris-HCI.
pH 8.0,
I mM EDTA,
6 M urea at a flow rate of 0.3 ml/min.
Elution
was accomplished
with a gradient
of 1 mM
NaC’l/ml
buffer made by mixing
buffer A (containing
0.65 M NaCI) with buffer B (with 0.76 M NaCl).
DISCUSSION
Applications
Some examples of the use of Mono Q and
Mono P in different separations are illustrated in Figs. 15- 17.
The columns used in this investigation are
designed for protein separations based on
A254
, N&l
0.04
I500
1000
1.500
DNA Sk.2 bp)
FIG. 9. Ionic strength of different chloride salts needed
for elution of DNA fragments
from the Mono Q column. Twenty
micrograms
of the standard
sample was
separated in 20 mM Tris-HCI.
1 mM EDTA, pH 8.3. at a
flow rate of0.5 ml/mitt.
The DNA was eluted with linear
gradients
(2.8 mM/mI)
of the different
salts. The ionic
concentration
in the peaks of some selected fragments
was measured and plotted in the figure.
20
10
5
30
40
10
ml
h
FIG. I 1. Rechromatography
of the 506-bp fragment
from Fig. 5a. The volume of the pooled peak was 4 ml.
After dilution
to 5 ml with water the pooled material was
applied directly
to the column
by 10 repetitive
injections. Flow rate during sample application
was 1 ml/
min. The chromatographic
conditions
were exactly as in
Fig. 5a, except during the first part of the gradient.
The
position, shape. and area ofthe eluted peak can therefore
be compared
with the corresponding
peak in Fig. 5a.
The yield was in this case calculated
as 93%.
ION-EXCHANGE
SEPARATION
OF
RESTRICTION
FRAGMENTS
165
i
FIG. 12. Large-scale separation
of Ifinfl-digested
pBR322 on Mono Q (a) and Mono P (b). In (a) 190 fig
of DNA in I ml of 20 mM Tris-HCI.
pH 8.3. was applied and the DNA was eluted with a linear gradient of
NaCI, I .3 mrd/ml,
made by mixing buffer A (containing
0.5 M of NaCI) with buffer B (containing
I .O M of
NaCI). Flow rate was 0.15 ml/min.
In (b) 200 tig ofthe same sample was separated in 20 mM Tris-HCl,
pH
8.0, at the same flow rate and gradient slope as in (a). Gradient
was made by mixing buffer A (containing
0.72 M of NaCI) with buffer B (containing
0.84 M of NaCI).
two different chromatographic
techniques.
The Mono Q column contains quaternary
amino groups for conventional ion-exchange
chromatography.
This column thus has the
same charge density over the whole pH
range. Mono P, on the other hand, is designed for chromatofocusing
and contains a
wide spectrum
of amino groups with pK
values evenly spread over the whole pH
range. On this column each sample molecule
will interact with a number of the different
amino groups. Furthermore,
the net charge
of the matrix will be strongly pH dependent,
highly charged at low pH and little charged at
high pH. In chromatofocusing
of proteins
the elution is accomplished with a decreasing
pH gradient obtained by titrating the gel with
special buffer mixtures
known
as polybuffers. This chromatographic
technique is
not applicable to nucleic acids for two reasons: (i) Nucleic acids do not have isoelectric
points in the working range of the chromatofocusing system and (ii) the nucleic acids
form strong complexes with the polybuffer
substances, giving rise to all kinds of artifactual separations.
In this investigation
we therefore used
both columns for conventional ion-exchange
chromatography.
In fact, by comparing the
results of the Mono P and the Mono Q col-
166
WESTMAN
1’
t1
&254
l&I
3.016
1.0
M
1,
1631
0.5
20
L
2
40
3
4
ml
I
5
6
h
FIG. 13. Separation
of 2.5 pg Hinfl-digested
pBR322
on Mono Q. Chromatographic
conditions
were the same
as in Fig. 12a. except for the gradient slope. which was
2.8 mM/ml.
and the sensitivity
setting of the detector.
umns we had an opportunity to investigate
the effect of charge density of the medium on
chromatographic behavior.
ET AL
This allows for extreme flexibility
in the
choice of chromatographic
buffer. With
Mono P the separations at pH 3.8 and 10.0
were practically identical with the separations obtained in the neutral pH range, despite the fact that at pH 3.8 the Mono P is
highly charged while at pH 10.0 it is hardly
charged at all. Beside the different concentration of the salt gradient the most significant difference is that for a given gradient
slope the chromatograms
are slightly more
compressed at high pH (Figs. 2b and 3)
probably reflecting less-tight binding to the
less-charged matrix. The remarkable separation range on Mono P in terms of buffer pH
as illustrated by Fig. 3 can be taken advantage of in case a particular buffer or buffer
pH is desired or, if there is an advantage, can
elute the DNA at a specific salt concentration.
Flow Rate
Not surprisingly
it was found that the
lower the flow rate the better the resolution
for all practically useful flow rates (Figs.
On Mono Q the separations were remark4-6). Less expected was the finding that
ably insensitive to buffer pH and composiMono Q seems to give a higher resolution of
tion, at least between pH 6 and 9 (Fig. 1). smaller fragments and Mono P a higher reso-
,163l
-1000
631
FIG. 14. The I63 I -bp peak from the separation
in Fig. I2a was collected. Part of the material was treated
with EcoRl and the reaction products were analyzed by electrophoresis
and chromatography
on Mono Q.
Chromatographic
conditions
were essentially the same as in Fig. 12a.
ION-EXCHANGE
SEPARATION
OF
RESTRICTION
FRAGMENTS
167
FIG. 15. Separation
of 30 qg HadlI-digested
pBR322 on Mono Q in 20 mM Tris-HCI,
pH 8.3. Flow rate
was 0.15 ml;min.
Elution with NaCl gradient was obtained
by mixing buffer A (containing
0.4 M NaCl)
with buffer 13 (containing
1.0 M NaCl). Gradient
slope was 2.8 mM/ml.
The separated fragments
were
analyzed
by polyacrylamide
gradient gel electrophoresis.
lution of larger fragments (Fig. 6). However,
these calculations did not take into account
peaks on the
the distorted, “club-footed”
Mono P. We do not know with certainty the
t
2
h
FIG. 16. Analytical
separation
on Mono
P of two
DNA fragments
(790 and 1003 bp) cloned in pBR322.
After cloning and purification
by conventional
methods
the inserts were excised and purified by chromatography
on a 2 x 40-cm column of Sephacryl
S-500. The inserts
were well separated both from the plasmid (3672 bp) and
the low-molecular-weight
contaminants
such as nucleotides but were only partially
resolved from each other.
This separation
was accomplished
on a Mono P column
in 20 mM Tris-HCI,
pH 8.0. at a flow rate of0.3 ml/min.
The inserts were eluted with a NaCl gradient of I mM/ml
obtained
by mixing buffer A (containing
0.76 M NaCl)
with buffer B (containing
0.82 M NaCI).
reasons for these distortions except that they
represent chromatographic
artifacts: Rerunning a symmetrical part of a peak produces a
new club-footed peak, and they do not appear when the same sample is run on Mono
Q. They do seem to be related to the column
design, since shortening the Mono P column
to the same length as the Mono Q column
eliminated them completely.
For good separations it is necessary to use
rather shallow gradients since all DNA elutes
within a narrow concentration range (Figs. 7
and 8). This is perhaps not so surprising since
all DNA molecules have the same charge
density. Fortunately the elution position is
independent of gradient slope. A first crude
separation with a steep gradient can therefore be used to find the working range for the
more sophisticated separation.
During this study we noticed that the way
these shallow gradients at a high salt concentration are formed is very important. If the
difference in salt concentration
between
buffers A and B is too big, chromatographic
artifacts will appear: Monocomponent
peaks
will start to “separate” into subpeaks. (It is
for this reason that we, in most figure legends, give the salt concentration of the A and
B buffers.) Increased efficiency in gradient
168
WESTMAN
FIG. 17. Purification
performed
on a Mono
gradient of 2.8 mM/ml,
was 0.15 ml/min.
Composition
AL.
of a 380-bp insert from pBR322 cloned in Esckri~hia
crrli. The separation
was
Q column in 20 mM Tri-HCI,
I mM EDTA, pH 8.3. DNA was eluted with a NaCl
except for the beginning
where the gradient slope was much steeper. The flow rate
mixing can also be accomplished by having
two gradient mixers in series.
Compared to HPLC and FPLC of proteins, the separation of nucleic acids by ionexchange chromatography requires remarkably long running times. However, from
Figs. 4b, 5b, and 8 it is obvious that reasonably fast separations can be obtained with
only limited loss in resolution if steep gradients and “high” flow rates are used.
Gradient
ET
the concentration needed for elution is high
enough to precipitate the DNA.
Additives
All the additives investigated, urea (6 M),
formamide (up to 2 M), acetonitrile (20%).
and EDTA and Mg’+ in millimolar
concentrations, had a very small effect on the separations, so there seems to be little limitation
of the additives that can be used.
A surprising finding illustrated in Fig. 10 is
that very shallow gradients are required in
urea to get spacing between the peaks.
A very common additive in nucleic acid
work is EDTA, which is added to protect the
sample from nuclease digestion. Although
EDTA in no way seems to interfere with the
separation, we still prefer to perform the separations without EDTA whenever possible.
EDTA often contains impurities which elute
as extra peaks. This is of course especially
prominent when very small amounts of sample are chromatographed.
Similar problems
with EDTA have been reported earlier (24).
Very similar separations were obtained
with all the monovalent cations investigated.
Multivalent
cations were not investigated.
From Fig. 9 it is clear that smaller cations are
much more effective as eluting agents than
larger cations. This probably reflects tighter
binding of counterions with a higher charge
density and thus a more effective neutralization of the DNA.
Why chaotropic anions such as acetate
and, to an even higher degree, trichloroacetate have such detrimental effects on the separation we do not understand, since other
Sample Working Range
denaturing agents such as urea, formamide,
and acetonitrile have very small effects (see
The working range in terms of amount of
below). With sodium sulfate we suspect that sample is illustrated in Figs. 12 and 13. Two
ION-EXCHANGE
SEPARATION
hundred micrograms of Hinfl-digested DNA
(Figs. 12a and b) correspond to 74 pg in the
163 1-bp peak. Compare this with the 75-bp
peak in the 2.5-pg experiment
(Fig. 13).
which contains about 42 ng DNA: a quantity
difference of a factor of almost 2000. The
ultimate limits are yet to be determined.
A strong advantage with Mono P and
Mono Q chromatography is the high yields.
Generally recoveries exceed 90% which we
believe is unmatched by any other DNA separation procedure, especially electrophoretic
procedures. where typical yields are below
50%. Nor have we been able to detect any
detrimental
effects on the quality of the
DNA by the chromatographic
procedure
(Fig. 14).
On
the Separation Mechanism
By performing separations on Mono P at
high pH where the column has a very low
charge content and at low pH where the column is highly charged and comparing these
results with results from Mono Q, which
always has the same charge content, WC expected to get some indications as to whether
the column should have a high or low charge
content to give the best resolution. To our
surprise it turned out that charge content
seemed to matter very little. Nor did it seem
to matter that the ionic groups on Mono P
are far from well defined. The common belief that an ion-exchange matrix should have
well-defined ionic substituents for optima1
separations seems not to be true, at least for
DNA fragments.
As for the separation principle, it is quite
obvious that straightforward ion exchange is
the predominant
factor. However, a closer
look at the chromatograms reveals that ion
exchange is not the only factor of importance. By looking at, for example, Figs. 1 and
2, and comparing the separation between
fragments 3441396 and 506/5 17. respectively, it is obvious that the separation be-
OF
RESTRICTION
FRAGMENTS
169
tween the 506 and 5 17 fragments is “too
good.” This becomes even more obvious if a
plot of elution volume or salt concentration
versus fragment size is made. By doing this
comparison and several others we have concluded that these discrepancies are due to
differences in overall base composition.
Fragments with a relatively high AT content
are retarded more than others. For example,
the AT content of the fragments mentioned
above are 344 bp =I 42%. 396 bp = 43%, 506
bp = 49% and 51’7-bp = 56%. The relative
distance between the 344- and the 396-bp
fragments is thus “normal”
while the distance between the 506- and 5 17-bp fragments is increased by the extra retardation of
the AT-rich 5 17-bp fragment. Sometimes
elution order may even be reversed (see, for
instance, the 458-bp fragment in Fig. 15).
A plot of elution salt concentration versus
fragment size will thus show kinks depending
on differences in AT content. This is especially prominent with fragments larger than
ca. 300 bp. The migration
of the smaller
fragments seems to be more closely regulated
by fragment size than by AT content. However if a normalized, apparent fragment size
according to the formula apparent fragment
size (bp) = true fragment size (bp) X concn
AT (%)/concn GC (%) is used in the plot,
relatively smooth curves will be obtained
even for the larger fragments (Fig. 18). The
AT influence on elution position was verified
also by data from Ref. (17) by plotting according to Fig. 18 (not shown). This effect
can be used for estimation of AT content if
the true fragment size is known.
The physicochemical reason why AT pairs
should interact more strongly with the column than GC pairs is still obscure to us. It is
not likely to be due to hydrophobic interaction since the same effect is observed in 6 M
urea as in 20% acetonitrile. Nor do we find
hydrogen-bond types of interactions to be a
likely cause: The effect is equally strong at
pH values as extreme as pH 3.9 and 10.0.
where the hydrogen bond interactions
should be completely different. Neither have
170
WESTMAN
DNA
pBr 322 Hmf I Mono P
Size
(Inz)
ET AL.
DNA size
(In Norm
PB~ 322 HAE
m Mono
0
bd
0
(In bp)
A
(In Norm
bp)
0
7 8
I
I
FIG. 18. Plot of true and normalized
fragment
size as a function
of elution volume for Hinfl fragments
from pBR322 separated on Mono P (a) and Hue111 fragments
separated on Mono Q (b). Fragment
size is
expressed as the natural logarithm
of the number of base pairs. “Normalization”
according to AT content
was done according
to the formula given in the text. Normalized
lengths are given within parentheses.
we been able to detect any correlation with
the base content in the sticky ends of the
fragments.
Delayed elution of AT-rich fragments was
observed also on RPC-5 columns. On these
columns with their mixed-mode
function
even more explanations for this behavior are
possible (25).
The more effective elution with smaller
cations indicates that elution is probably due
partly to decreased net charge on the DNA
by the counterions and partly to competitions between eluting anion and DNA for
charged groups on the column. Small ions
with high charge density will bind more
strongly to the DNA than larger ions.
In conclusion we can say that ion-exchange chromatography on Mono Q as well
as Mono P is a powerful tool for DNA fragment separation. The technique is very easy
to set up and use since it is extremely tolerant
to chromatographic
conditions: Buffer type
and pH, flow rate, gradient slope and composition, and additives can be varied between very wide limits. Which column to
choose is almost a matter of taste: Mono Q
gives more symmetrical peaks than Mono P
and also gives somewhat better separation of
smaller fragments. Mono P on the other
hand, can tolerate higher flow rate than
Mono Q and separates the larger fragments
somewhat better. The major drawbacks with
Mono P are the distorted peaks and the pH
sensitivity. The distorted peaks may give rise
to cross-contamination
in preparative separations and the pH sensitivity makes careful
buffer preparation essential for the gradient
not to fall out of the separation range. On the
other hand, the pH sensitivity provides a
means for modifying the composition of the
eluting buffer so that the purified DNA can
be recovered in any desired milieu.
ACKNOWLEDGMENT
We thank Dr. Bengt
comments
and linquistic
osterlund,
revision.
Pharmacia
AB,
for
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