Download Sheared DNA fragment sizing: comparison of techniques

Volume 3 no.11 November 1976
Nucleic Acids Research
Sheared DNA fragment sizing: comparison of techniques
Charles P.Ordahl, Thomas R.Johnson and Arnold I. Caplan
Departments of Biology and Anatomy, Case Western Reserve University,
Cleveland, OH 44106, USA
Received 2 August 1976
ABSTRACT
DNA fragmented by conventional French press shearing procedures (30,000
lbs/in^) has a number-average fragment size of 230 base pairs. This is considerably smaller than the 450 base pairs typically reported for DNA sheared
by this method. Comparison of 5 sizing techniques indicates that sheared DNA
fragment size is overestimated by either measurement of velocity sedimentation
or Kleinschmldt Electron Microscopic visualization. Both adsorption grid
electron microscopic visualization and gel electrophoresis yield the most reliable estimates of the mean size of small DNA fragment populations. In addition, the assessment of fragment size distribution (not possible from sedimentation analysis) potentially allows more critical evaluation of DNA hybridization and reassociation kinetic and measurement parameters.
INTRODUCTION
DNA reassociation and hybridization analysis requires the use of fragmented DNA . Precise information regarding the size of the DNA fragments is
essential to interpretation of these kinds of experiments.
Fragment size is
not only an integral element in the kinetics of DNA reassociation and hybridization, but also affects the kinds of DNA-DNA or DNA-RNA duplexes obtained.
For some experiments it is necessary to use long (>1000 base pair) fragments
of DNA. Fragments of this size have been used to determine the extent of inter2 3 4
sperslon of repeated and nonrepeated sequences of DNA in eucaryotes ' '
In general, however, on kinetic and other grounds it is advantageous to use
short DNA fragments less than 500 base pairs in length . This length not
only permits separation of the repetitive and nonrepetitive components of most
eucaryotlc genomes ' ' , but also reduces the amount of single strand tails
7 8 9
in reassociated and hybrid duplexes ' '
A variety of methods have been employed to shear high molecular weight
DNA to fragments of a particular desired size . One of the most common
methods is to force a DNA solution through the valve of a high pressure cell
2 5
at a constant pressure of between 30,000 and 50,000 lbs/in
.
DNA frag-
mented in this manner i s typically reported to be 450 base pairs in length
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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1 2
3 5 9
as determined from measurement of sedimentation velocity ' ' ' '
results presented here show that DNA sheared at 30,000 lbs/in
ably smaller than the 450 base pairs typically reported.
The
is consider-
DNA sheared in
this manner has a mean fragment length of 220-235 base pairs as determined by
gel electrophoresis and adsorption grid electron microscopy.
Comparison of
5 sizing techniques indicates that both velocity sedimentation and Kleinschmidt electron microscopy overestimate mean fragment size in sheared DNA
preparations.
The implications of these observations are discussed as they
relate to the current reassociation and hybridization technology.
METHODS AND MATERIALS
Preparation of DNA
Nuclei were prepared by homogenizing chick embryonic brain tissue in
0.075 M NaCl, 0.025 M EDTA, pH 8 (Buffer A) at 4°C with 1% NP-40 (Shell) in
a loose fitting dounce homogenizer.
Cell breakage was virtually complete and
nuclei were pelleted at 3,000 RPM for 10 min (Sorvall HB-4) and resuspended
in Buffer A without NP-40 using a dounce homogenizer.
The nuclei were again
pelleted and resuspended in 0.1 M NaCl, 0.05 M Tris pH 7.4, 0.001 M EDTA
(Buffer B ) ; SDS was added to a final concentration of 1% and the suspension
homogenized.
An equal volume of phenol saturated in Buffer B was added and
the mixture shaken at room temperature for 20 minutes and then centrifuged
at 5,000 RPM for 10 min at 20°C.
The aqueous phase was removed and extracted
twice more with phenol as above and then extracted once with chloroform:
octanol (8:1) to remove phenol and precipitated overnight at -20°C with 2.5
volumes of ethanol.
The DNA precipitate was pelleted at 10,000 RPM for 30
min at -10°C and redissolved in Buffer B at a concentration of <5 mg/ml
(A 2 g. = 100). This solution was then loaded into a french pressure cell
(Aminco) and expelled at a constant pressure drop of 30,000 psi using an
Aminco french press at a rate of 1-5 mls/min.
Sheared DNA was then treated
with RNase B (Worthington 50 yg/ml 1 hr 37°C) and pronase (Sigma 50 ug/ml
1 hr 37°C) and phenol extracted and ethanol precipitated as above.
After
collecting the ethanol precipitated DNA, it was excluded on Sephadex G-100
(Pharmacia) to remove digested RNA fragments and phenol and stored as an
ethanol precipitate at -20°C until needed.
A
A
manner had an 260^ 280
=
1-83-1.85.
bias
nic fibroblast culture using
10
previously
Sheared DNA purified in this
DNA was radiolabeled in chick embryo-
H-Thymidine (Amersham Searle) as described
Sucrose Gradient Centrifugation
Neutral linear 5-20% sucrose gradients were made using Buffer B in SW-
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40 Ti (Beckman) cellulose nitrate tubes (final volume 13 mis).
DNA dissolved
in 200 yl of Buffer B was layered onto the top and the gradients centrifuged
at 30,000 RPM for 16 hrs at 4°C.
Alkaline sucrose gradients were identical
except that sucrose gradients were prepared in 0.1 M NaOH (pH 13).
The DNA
was dissolved in 0.1 M NaOH and heated to 65°C for 5 min before layering
onto gradients.
Centrifugation conditions were identical to those of the
neutral gradients.
Gradients were analyzed for absorbance at 254 nm on an
Isco Model UA-4 gradient fractionator and recorder.
Fractions (0.375 ml)
were collected and neutralized with HC1 where appropriate.
Radioactively
labeled DNA was quantitated in trlton based scintillation cocktail (33% V/V
Triton, 66% V/V Toluene, 0.53% W/V Omnifluor [Beckman]) at 30% efficiency.
Model E Centrifugation
Sedimentation analysis was conducted in a Beckman-Spinco Model E analytical ultracentrifuge equipped with an RTIC unit, electronic speed control,
photoelectric scanner and multiplexer.
An AnF rotor was used which incorpo-
rated 3 cells with 12 mm light paths for scanning at 260 nm with ultraviolet
optics.
Centrifugation was conducted at 52,000 RPM at 20°C.
DNA was sedi-
mented in 0.12 M sodium phosphate buffer (NaPB pH 6.8). Observed S values
were corrected to S-Q, W in the manner described by Studier
after deter-
mining the density (1.0114 g/cc) and viscosity relative to water (1.044) of
0.12 M sodium phosphate buffer.
Sedimentation coefficients were determined
by the method of midpoints for all runs.
The log of the distance of the
sedimenting boundary from the axis of rotation at the half-height of the
boundary was plotted against time (seconds) and the sedimentation coefficient
calculated from the slope of the line of best fit.
Electron Microscopy
For the grid adsorption method, DNA was mounted on grids from 0.12 M
phosphate buffer and stained exactly as described in Johnson & Caston
Kleinschmidt spreadings (13) were performed using a distilled water hypophase.
The final composition of the hyperphase was:
DNA, about 2 ug/ml;
0.02 M Tris, pH 7; 1 M ammonium acetate; and 0.01% cytochrome c.
Films were
picked up on carbon-coated Colloidon films and contrasted by shadowing with
Pt:Pd 80-20.
SV40 DNA (m.w. = 3.1 x 10 6 daltons) (kindly provided by Dr.
Peter Tegtmeyer) was included for both procedures to serve as a length
standard.
For the grid adsorption method, micrographs were taken at a magni-
fication of 60,000 to 80,000; for Kleinschmidt preparations, at about 20,000.
Microscopy was performed with a Siemens 101A, operated at 80kV.
Magnifica-
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tions were calibrated with a diffraction grating replica (58,000 lines/inch;
Polysciences).
Disc Gel Electrophoresis
Disc gel electrophoresis was performed essentially by the method of
Carroll & Brown1 . Disc gels (2.4% acrylamide, .12% bisacrylamide, 0.5%
Agarose; 200 mm) were run at 5mAMP/tube for 4-5 hrs.
Hind III nuclease (Miles)
fragments of lambda DNA (Miles) was prepared according to Danna, Sack and
Nathans
. After electrophoresis the gels were stained for 30 minutes in
5 ug/ml ethidium bromide and then were irradiated on a long wave ultraviolet
light box and photographed on Polaroid transparencies
. The migration dis-
tances were measured on photographic prints (see Figure 5 ) .
Other gels were
scanned at 260nm on a Gilford linear transport gel scanner to quantitate and
record the distribution of sheared DNA fragments in the gel.
The correspond-
ing position of the Hind III fragments of lambda DNA was plotted on the
recording (as in Figure 5) to calibrate the scan for distance versus molecular weight.
The calibrated recording was then divided into increments of 25
base pairs and the area under the ultraviolet profile at each increment cut
out and weighed on an analytical balance.
The weight is directly proportional
to the area and is therefore a measure of the relative mass of DNA at each 25
base pair increment.
4, panel "f".
The results of these determinations is shown in Figure
To determine the relative number of DNA molecules at each gel
position (Figure 4, panel " c " ) , the relative absorbence was divided by the
calibrated number of base pairs (i.e., rel. ^QQIIIO.
fragments).
base pairs = rel. no.
Thus, taking a given absorbence reading at the 200 base pair
position of the gel as corresponding to x number of DNA fragments, then the
same absorbence reading at the 100 base pair position would correspond to 2x
number of DNA fragments.
The molecular weights of the fragments of lambda DNA generated by Hind
III nuclease fragments were determined by relative electrophoretic mobility
and by electron microscopy.
reported by Carroll and Brown
The fragment lengths are identical to those
except for the smallest fragment (VII) which
was found to be 395 base pairs in length rather than 525 base pairs in
length.
The differences in fragment length probably reflect differences
between the lambda DNA obtained from Miles Laboratories and that employed by
Carroll and Brown rather than differences in measurement technique or
accuracy.
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RESULTS
I.
Sedimentation Analysis
Historically, the most commonly used method for sizing DNA has been
sucrose gradient centrifugation which was used to estimate sedimentation coefficient (S value).
Figure 1 shows the sedimentation of sheared native
(undenatured) DNA relative to that of ribosomal RNA in a linear, neutral
sucrose gradient.
This sizing approach is advantageous in that it is rapid
and readily accessible to most laboratories and it affords an approximation
of the average size and homogeneity of DNA fragments.
Under denaturing (0.1
M NaOH) conditions there is no broadening of the sheared DNA peak indicating
that there are few, if any^ "cryptic" single strand sissions in the sheared
native DNA fragments.
The sharpness of the sheared DNA peak is misleading
28s
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10
20
FRACTION
30
Fig. 1 Sucrose Gradient Sedimentation of Sheared DNA. Solid line shows the
absorbtion trace of sheared DNA sedimentation in a neutral 5-20% sucrose
gradient. Inclusion of 3H-labeled DNA (closed circles) demonstrated that
background absorbence at the tube bottom is not due to DNA. The broken line
shows the absorbence tracing for ribosomal RNA sedimentation in a parallel
gradient. For procedural details see Methods and Materials.
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because, as demonstrated below, there is considerable heterogeneity in fragment size.
Comparing the rate of sedimentation of the sheared native DNA to that of
the 5S, 18S and 28S ribosomal RNA species yields an approximate sedimentation
coefficient of 7S.
Using the Studier equation
relating the sedimentation
coefficient [S] to molecular weight [M] of native DNA under nondenaturing
conditions;
S = 0.0882 M
yields a value of M = 3.1 x 10
per fragment.
0>346
;
or an average of approximately 475 base pairs
As demonstrated below, this is a considerable overestimate of
the true average size of these DNA fragments.
This overestimate arises, in
part, from inaccurate measurement of S value by sucrose gradient sedimentation.
The measurement of S value can be made more accurately by direct
measurement in Model E centrifugation.
S-Q
Figure 2 shows the change in value
for sheared DNA as measured in 8 Model E centrifugations over a 10-fold
concentration range.
Extrapolation of this change in S
tration yields a value of S.Q
20,w to zero concenof 6.7 which represents an estimate of the
sedimentation velocity of the sheared DNA in the absence of intermolecular
0.2
0.1
10
20
30
40
ug DNA/ml
Fig. 2 Sedimentation Analysis of Sheared DNA. Sheared DNA was sedimented
in 8 -model E centrifuge runs at various concentrations and the observed S
values corrected to S20 w as described in Methods and Materials. Extrapolation of the value l/S2o'w * 8 s n o w n a s a means of estimating the sedimentation
velocity in the absence'of intermolecular interaction (S°,Q w ) .
The value for
1 / S 2 Q w is 0.1475 corresponding to S§o,w = 6.7S.
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effects.
Using the Studier equation, as above, this S value estimates the
sheared DNA molecular weight to be 272,000 daltons or approximately 420 base
pairs.
It is clear that even slight inaccuracies in estimation of S can give
rise to large inaccuracies in molecular weight estimation.
Further experi-
ments presented below, however, also indicate that the Studier formula for
relating S to DNA molecular weight also tends to overestimate the molecular
weight of very small DNA fragments such as those being measured here (see
Discussion).
II.
Electron Microscopic Visualization
Visualization of DNA molecules in the electron microscope permits an
essentially direct measurement of fragment size.
This technique employs
fewer correction factors and assumptions necessary to translate measurement
into molecular weight.
The most common technique for visualizing DNA is the
Kleinschmidt procedure in which DNA embedded in a protein film is applied to
a grid and rendered electron dense by staining or metal evaporation
Figure 3a shows an electron micrograph of sheared native DNA as visualized by
the Kleinschmidt procedure and Figure 4a and d shows the distribution of
contour lengths (in base pairs) of 379 sheared native DNA fragments visualized by this technique.
The number average length of the DNA fragments in
this distribution is 333 base pairs.
•m
Fig. 3 Electron Micrographs of Sheared DNA. Panel "a" is a low magnification
of sheared DNA as visualized in a Kleinschmidt preparation. Circular DNA
molecule in "a" is SV-40 DNA included in all preparations as standard. Panel
"b" is higher magnification of sheared DNA visualized by the adsorption grid
procedure. The arrow in "b" indicates a 100 base pair fragment. Panel "c"
is Kleinschmidt grid visualized at the same magnification as in panel "b".
The bracketed bar spans 1000A in each panel.
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Figure 3b shows sheared DNA as visualized by the adsorption grid electron microscopic procedure.
This technique differs significantly from the
Kleinschmidt procedure in that visualization of the DNA is done by direct
staining without intervention of a coating of cytochrome c.
Briefly, DNA is
adsorbed onto grids charged with the commercial disinfectant Zephiran and
12
stained with uranyl acetate . As a result, the visualized DNA fragments
have a diameter of approximately 20A, closely approximating that expected for
DNA in solution, and the DNA fragments are easily resolved above grid background (Figure 3b). Comparison, at the same magnification, of an adsorption
grid micrograph (Figure 3b) to a Kleinschmidt micrograph (Figure 3c) demonstrates the increased resolution afforded by the grid adsoprtion procedure.
For example, the 100 base pair fragment indicated by the arrow in Figure 3b
CO
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
BASE PAIRS ( x i o o )
Fig. 4 Size Distribution of Sheared DNA Fragments. Panels a, b and c show
the relative number of DNA molecules corresponding to given length increments
(base pairs) as determined by Kleinschmidt EM, Adsorption EM and gel electrophoresis procedures, respectively. Panels d, e and f show the relative mass
of DNA at each length increment as determined respectively by the Kleinschmidt EM,Adsorption EM and gel electrophoresis procedures. The vertical
bar in a, b and c indicates the median fragment number. In d, e and f the
vertical bars labeled "n" represent the number average length while that
labeled "w" represents the weight average length. Relative number and mass
units were used to facilitate comparisons between the 3 measurement techniques.
The units for each panel were established by setting the arbitrary value of
10 at the measurement increment with the greatest abundance of DNA mass or
number. This makes all distribution profiles have the same maximum height.
The actual number of DNA fragments measured by electron microscopy was 379
in the Kleinschmidt preparation and 640 in the adsorption grid preparation.
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would be indistinguishable from grid background in Figure 3c.
It is not sur-
prising, therefore, that the distribution of fragment lengths as determined
by adsorption grid microscopy (Figures 4c and 4f) demonstrates the presence
of a large number of small (less than 100 base pair) DNA fragments which were
not scored by the Kleinschmidt procedure.
This, of course, has a considerable
effect on the estimates of average fragment size obtained by the two methods
(see Discussion).
III.
Gel Electrophoresis
Gel electrophoresis permits highly precise estimates of DNA fragment
size because DNA fragment standards of precisely known molecular weight are
available by digestion of viral DNA with restriction nuclease.
Figure 5 shows
that the relative electrophoretic mobility of lambda DNA fragments resulting
from Hind III nuclease digestion is linear with respect to the logarithm of
the base pair length for fragments less than 3,000 base pairs long.
These
restriction fragments therefore serve to calibrate the gel to relate DNA
electrophoretic mobility to DNA molecular weight and as a result a logarithmic
base pair scale can be superimposed over the gel length (Figure 5 ) .
Using
this scale it can be seen that the majority of the sheared DNA fragments
migrate at a rate indicating that they are between 200 and 300 base pairs in
length.
Examination of a gel as in Figure 5 does not, however, permit critical
evaluation of the distribution of fragment lengths because the base pair
scale is logarithmic and thereby tends to concentrate larger fragments and
dilute smaller fragments.
To assess the distribution of fragments, cali-
brated gels were scanned at 260 nm and the U.V. profile replotted on a linear
scale of fragment length (Figure 4f, and Methods and Materials).
Here it can
be seen that the mean fragment length (number average) is 233 base pairs and
that there is a predominant mode of fragments centered at approximately 200
base pairs.
The median fragment length is 200 base pairs as shown in Figure
6c by a plot of the same data in terms of the relative number of DNA fragments in each size class.
The data in Figure 6c and f demonstrate that an
electropherogram such as in Figure 5 does not readily permit assessment of
either the distribution or mean length of heterodisperse DNA fragment populations such as those obtained by shearing processes.
It is therefore advan-
tageous to replot gel electropherograms to assess the distribution of fragment size in heterodisperse DNA fragment populations.
This distribution
profile also permits more critical comparison of the relative accuracy of the
various sizing techniques studied (see Discussion).
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8
5
BASE PAIRS (x 100)+
9876 5 4
mi
3
i i i
If
'
J
'——t
'
Relative Migration
g
'—
Fig. 5 1 Electrophoretic Mobility of Sheared DNA Relative to Hind III DigestedLambda BNA. Bottom panel shows position of Hind Ill-Lambda DNA markers (Gel
A & C) relative to sheared DHA (Gel B) . Upper panel shows the line establishing the relationship between relative electrophoretic mobility of Hind III
digested lambda DNA fragments as standards and molecular weight in base pairs.
This line calibrates the gel permitting superimposition of the base pair scale
above the gels. Closed circles show position of Hind III lambda fragments.
Open circle shows apparent midpoint of sheared DNA band. Right angles along
calibration line show the relationship between the horizontal base pair scale
superimposed over the gels and base pair scale on the vertical axis. For
details see Methods and Materials.
DISCUSSION
The purpose of the measurements reported here is to compare the estimates of sheared DNA fragment size obtained by a variety of techniques.
The
results indicate that conventional French press shearing techniques (30,000
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9
lbs/in ) yields fragments approximately 230 base pairs in length.
In most
cases DNA fragmented by French press shearing is reported to be 450 base pairs
1 2
3 5 9
in length ' ' ' ' ,
as determined by sedimentation velocity analysis.
Comparison of the data obtained from the sizing techniques employed here indicates that the average size of sheared DNA fragment populations is overestimated by either velocity sedimentation of Kleinschmidt electron microscopic
analysis.
Comparisons of average molecular weight estimates obtained by different
techniques cannot always be made directly because some measurement techniques
give disproportionate weight to the largest molecules in a DNA fragment population
. This gives rise to different estimates of average molecular weight
which are not related to measurement accuracy.
Table I compares the esti-
mates of sheared DNA fragment size which are obtained from each technique as
they relate to weight average molecular and median molecular weight.
velocity sedimentation only gives a weight average molecular weight
Because
, it is
appropriate to compare this technique to the others only with respect to this
value.
Even when this is done, it can be seen that Model E centrifugation
determination of S value gives an estimate of weight average molecular weight
approximately 30% higher than that obtained by either gel electrophoresis or
adsorption grid electron microscopy (Table I ) . While there is apparent
Table I.
Estimates of Average Molecular Weight in Base Pairs of Sheared DNA;*
Measurement Technique
Median
Weight
Mw
Sucrose Gradient Centrifugation
475
Model E Centrifugation
420
Mn
Median
Number
Kleinschmidt EM
360
393
333
295
Adsorption Grid EM
278
293
222
204
Gel Electrophoresis
260
312
233
222
* Molecular weight averages, expressed in base pairs, were calculated by the
following formulas:
£ n-j M-i
M.T = „ 7 .,1
w
Z n^_ M-;
.
; and
Where n^ is the number and M.^ is the base pair length of the DNA fragments at
each measurement, ^ is the weight average molecular weight and M^ is the
number average molecular weight^?. Median weight and median number show the
midpoint of the fragment populations as assessed from total fragment weight
and number, respectively. Sucrose gradient and Model E centrifugation estimates were made using Studier equation^-'-. All other estimates were calculated from the data shown in Figure 5.
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reasonable agreement between the Model E values and the Kleinschmidt electron
microscopy value,other considerations, presented below, indicate the latter
technique also overestimates the average size of heterodisperse populations
of small DNA fragments.
We conclude, therefore, that estimation of the
molecular weight of small DNA fragments by either sucrose gradient or Model E
centrifugation overestimates the true weight average size of the fragments.
This is most probably due to the fact that Studier used DNA molecules over
100 times larger than those being measured here to formulate the equations
relating S value to molecular weight for DNA
.
It would appear possible
to re-calibrate the relationship between S and molecular weight for small DNA
fragments using restriction endonuclease DNA fragments of known molecular
weight.
However, even if this were done, the use of sedimentation velocity
measurement, although rapid and convenient, would remain less satisfactory
for measuring small DNA fragments than the other methods discussed below
because it is limited to estimation of weight average molecular weight alone.
Because this average is strongly biased towards the molecules of highest
molecular weight
, it is less useful for calculation of the kinetic para-
meters involved in DNA hybridization and reassociation as discussed below.
The more useful number average molecular weight estimate is impossible to
derive from the weight average without knowing the distribution of fragment
lengths.
Thus, even under ideal circumstances, the estimation of the average
molecular weight of heterodisperse DNA fragment populations by sedimentation
velocity is potentially less useful than estimates obtained by the other
techniques described below.
The use of electron microscopy to measure fragment size is, in general,
a satisfactory method for measuring DNA fragment sizes. Both fragment length
and number are directly measured and as a result reliable size distributions
are obtained.
This reliability depends upon first, measuring large numbers
of fragments which is a laborious process; and second, insuring that no size
bias is incurred during the measurement process which would tend to misrepresent the distribution.
Two different electron microscopic sizing procedures
gave significantly different size distributions (Figure 4) and molecular
weight averages (Table I) for the sheared DNA fragments.
Comparison of the
fragment distribution obtained by the two methods (Figure 4) reveals that
less than 18% of the Kleinschmidt visualized fragments are shorter than 200
base pairs as compared to 47% as visualized by the adsorption grid method.
This indicates that DNA fragments shorter than 200 base pairs are visualized
less effectively by the Kleinschmidt procedure and that fragments shorter
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than 100 base pairs are not resolved at all.
This is visually illustrated in
Figure 3b and 3c which compares an adsorption grid micrograph and a Kleinschmidt grid micrograph of sheared DNA at the same magnification.
The 100
base pair fragment seen in the adsorption grid micrograph (Figure 3b, arrow)
would obviously be difficult, or impossible, to visualize in the Kleinschmidt
micrograph (Figure 3c). The fact that the smaller molecules are not visualized in Kleinschmidt preparations undoubtedly results from their being obscured,
relative to grid background, by the coating of cytochrome f: and contrasting
material.
The size distribution in the two preparations are, however, remark-
ably similar for fragments longer than 200 base pairs (Figure 4 ) .
Thus, the
higher average size estimates obtained by the Kleinschmidt procedure are
entirely explained by the failure of this procedure to score the true relative proportion of fragments shorter than 200 base pairs.
We conclude, there-
fore, that a more reliable DNA fragment size distribution and more accurate
estimates of molecular weight averages are obtained by the adsorption grid
method of electron microscopy.
Gel electrophoresis is probably the most satisfactory method for sizing
heterodisperse DNA fragment populations because it is rapid, easily calibrated with restriction fragment standards, and permits estimation of the
distribution of fragment size.
Figure 4 shows the electropherogram of sheared
DNA on a gel calibrated with Hind III restriction nuclease fragments of lambda
DNA.
The sheared DNA migrates as a broad band the center of which is at a
position corresponding to approximately 250 base pairs with extremes at
approximately 400 base pairs and approximately 150 base pairs.
This apparent
distribution, however, is misleading because the correspondence between
molecular weight and distance migrated is semilogarlthmic.
To obtain a more
meaningful distribution profile, a calibrated gel was scanned for A,gQ run to
measure the relative amount of DNA throughout the gel and then the area under
each 25 base pair increment of the optical density profile was determined.
The results of this are shown in Figure 4f and in Figure 4c the relative number of DNA fragments at each increment is shown.
On a linear scale the
pattern of fragment length distribution is more readily assessed and further
permits computation of the various estimates of average molecular weights
shown in Table I.
Both the distribution of fragment lengths (Figure 4) and
the average molecular weight estimates (Table I) obtained by either gel
electrophoresis or by adsorption grid electron microscopy are essentially
identical.
These data therefore mutually support the conclusion that the
number average molecular weight (M ) of DNA sheared by conventional French
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press techniques is approximately 230 base pairs.
In addition, it is most
likely that previous reports that DNA sheared in this manner is 450 base
pairs in length result from estimation of weight average molecular weight
(Mw) and from inaccuracies relating S to molecular weight.
Both the weight average (Mw) and number average (Mft) molecular weight
are useful for computations involved with DNA reassociation and hybridization.
The knowledge of both averages may prove useful in understanding the differences between the expected and observed rates of DNA hybridization or reassociation.
For example, because the ratio of weight average molecular weight
to number average molecular weight is 1.33, during DNA reassociation at the
observed Cotl (Cot value at which 50% of the DNA mass is scored as duplex)
one would expect approximately 67% of the DNA fragments to be in reassociated
duplex.
Since it is fragment number which kinetically drives the reassocia-
tion of DNA, kinetic computations should ideally derive the concentration
terms from M
rather than M . Thus, it is potentially of great advantage in
most cases to determine the distribution of sheared DNA fragment sizes in
order to evaluate M
and M .
ACKNOWLEDGEMENTS
We would especially like to thank Kenneth Neet and Chuck Starling for
providing the Model E Centrifuge and the expertise to operate it.
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