Large DNA fragment sizing by flow cytometry: application to the characterization
of P1 artificial chromosome (PAC) clones
Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones
Zhengping
Huang
,
Jeffrey T.
Petty
+
,
Brian
O'Quinn
+
,
Jonathan L.
Longmire
1
,
Nancy C.
Brown
1
,
James H.
Jett
1
and
Richard A.
Keller*
Chemical Science and Technology Division and
1
Life Sciences Division, Los Alamos National Laboratory,
Los Alamos
, NM 87545,
USA
Received July 9, 1996;
Revised and Accepted September 13, 1996
ABSTRACT
A flow cytometry-based, ultrasensitive fluorescence detection technique is used to size
individual DNA fragments up to 167 kb in length. Application of this technology
to the sizing of P1 artificial chromosomes (PACs) in both linear and
supercoiled forms is described. It is demonstrated that this method is well
suited to characterizing PAC/BAC clones and will be very useful for the
analysis of large insert libraries. Fluorescence bursts are recorded as
individual, dye stained DNA fragments pass through a low power, focused,
continuous laser beam. The magnitudes of the fluorescence bursts are linearly
proportional to the lengths of the DNA fragments. The histograms of the burst
sizes are generated in <3 min with <1 pg of DNA. Results on linear fragments are consistent with those obtained by
pulsed-field gel electrophoresis. In comparison with pulsed-field gel electrophoresis, sizing of large DNA fragments by this
approach is more accurate, much faster, requires much less DNA, and is
independent of the DNA conformation.
INTRODUCTION
A central role in all physical genetic analyses is the sizing of DNA molecules,
which is typically accomplished using gel electrophoresis. Conventional
acrylamide and agarose gels have been used to separate DNA of lengths 10-2000 and 100-40 000 base pairs (bp) respectively (
1
). For DNA fragments larger than 40 kilobase pairs (kb), migration through the
gel is independent of the fragment length (
2
), so pulsed field gel electrophoresis (PFGE) is used (
3
). Although pulsed field electrophoresis adequately separates large DNA
molecules, to achieve high resolution separation requires tens of hours; gel-to-gel, day-to-day reproducibility and accuracy remain problematic (
2
,
4
). Furthermore, PFGE cannot be used to size large supercoiled or open circular
DNA (
5
); samples must be linearized before analysis. An optical contour maximization
technique, based on analyzing the optical images of single DNA fragments, is an
alternative approach (
6
). This methodology is relatively easy and fast to implement, but the sample
fragment selection is arbitrary and many fragments need to be analyzed to
achieve an accuracy comparable with PFGE (
7
). The applicability of pulsed-field technology to capillary electrophoresis shows considerable promise
for the rapid separation of DNA fragments up to 1.6 * 10
6
bp (
8
,
9
).
We (
10
-
12
) and others (
13
,
14
) have developed a flow cytometric (FCM) method to size DNA. DNA fragments are
stained with bisintercalating dye TOTO-1 [a thiazole orange homodimer (
15
)], which binds to DNA in a stoichiometric manner. Individual stained DNA
fragments are passed through the laser illuminated detection volume (~10 pl), producing fluorescence photon bursts. The burst size is
proportional to the number of dye molecules bound to DNA, and thus is
proportional to the fragment length. The primary advantage of our technique is
short data collection time (<3 min), high sensitivity (<1 pg of DNA was analyzed) and linear response versus fragment size and number
of fragments. Our previous published work demonstrated a sizing range of 1.5-48.5 kb (
12
).
Our goal is to size larger DNA fragments faster and more accurately than can be
done by pulsed field gel electrophoresis. This could be applied in the
construction of a high resolution physical map for each of the human
chromosomes. Completion of the map requires the availability of comprehensive
libraries of DNA clones in appropriate vectors. Cosmids allow inserts of 30-45 kb (
16
); the bacteriophage P1 cloning system accepts inserts in the 70-100 kb range (
17
); P1-derived artificial chromosomes (PACs) allow inserts of 70-300 kb (
18
); bacterial artificial chromosomes (BACs) also accept inserts up to 300 kb (
19
); yeast artificial chromosomes (YACs) can propagate exogenous DNA in excess of
1 * 10
6
bp (
20
). Due to drawbacks associated with cosmids (comparatively small sizes,
instability of clones) and YACs (low transformation efficiency, excessive
presence of chimeric clones, difficulties in DNA manipulation) (
18
), PAC/BAC systems are gaining increasing popularity (
21
).
In this work, we demonstrate the extension of our sizing range up to 167 kb. A
linear relationship between fluorescence burst size and the length of DNA
standards was observed. FCM was successfully applied to size both linear and
supercoiled PAC clones. Our results for linear clone sizes are in agreement
with those obtained by PFGE. Clones from PAC libraries can be sized directly
and routinely.
MATERIALS AND METHODS
DNA
Bacteriophage [lambda] DNA was obtained from GIBCO BRL Life Technologies (Gaitherburg, MD);
Kpn
I digests of [lambda] DNA and Low Range PFG Markers were obtained from New England BioLabs
(Beverly, MA). Coliphage T4 and T5 DNA were obtained from Sigma Chemical Co.
(St Louis, MO). Cl-7, Cl-10, Cl-32 and Cl-56 are PAC clones obtained directly from a PAC library
constructed at Los Alamos.
PAC clones Cl-26 and Cl-29 were isolated from
Escherichia coli
host cells using a rapid alkaline lysis miniprep method (
18
). This is a modification of a standard Qiagen-Tip method which uses no organic extractions or columns.
Escherichia coli
cells contained clones that were transformed into the cells by electroporation
(
22
). Two ml cultures of cells were grown for 18 h at 37oC in Luria Broth supplemented with 30 [mu]g/ml kanamycin. For the final step, PAC DNA was ethanol precipitated
and resuspended in 40 [mu]l of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The procedure resulted in a recovery of 0.5-2 [mu]g of DNA. Extra care was used to minimize the amount of
contamination from
E.coli
genomic DNA, cellular protein and RNA, and to minimize the loss and shearing of
clone DNA. After the cells were lysed all pipeting was done with wide-bore or cut pipet tips.
Restriction digestion
Eag
I digestion is required to linearize the PAC DNA before PFGE can be used to size
clones. A volume of 11.5 [mu]l of TE buffer, 2 [mu]l of NEB 3 buffer (50 mM Tris-HCl, 10 mM MgCl
2
, 100 mM NaCl, 1 mM DTT, pH 7.9), 5 [mu]l of DNA sample and 1.5 [mu]l (15 U) of
Eag
I (New England BioLabs, Beverly, MA) were mixed gently and incubated for 3 h at
37oC. The mixture was then heated at 65oC for 15 min to inactivate the enzyme.
Sample preparation
The stock DNA solutions were stored at 4oC. TOTO-1 (Molecular Probes, Eugene, OR) was stored as a 1 mM solution in
DMSO at -20oC. A 1 * 10
-5
M solution of TOTO-1 was prepared by diluting 1 [mu]l of the stock in 99 [mu]l of TE. To size intact supercoiled PAC clones, 1 [mu]l of the DNA clone (~20-50 ng/[mu]l), 0.7 [mu]l of 10 ng/[mu]l [lambda], 0.4 [mu]l of 10 ng/[mu]l [lambda]
Kpn
I digest, 2 [mu]l of 25 ng/[mu]l T4 DNA, and 3.4 [mu]l of 1 * 10
-5
M TOTO-1 solution were added to 150 [mu]l of TE. This gives concentrations of 2 * 10
-7
M TOTO-1 and 0.5-0.7 ng/[mu]l DNA, corresponding to a base pair/dye molecular ratio between
3.6:1 and 5:1. Only [lambda] and T4 were added as markers when sizing the
Eag
I digest of PAC clones. The mixture solutions were incubated for at least 60 min
at room temperature in the dark and diluted 150-fold in TE to give a final total fragment concentration of ~5 * 10
-14
M. The final clone concentration was ~1.5 ng/ml. The DNA/dye complex was very stable; narrow burst distributions
were obtained for samples sitting at room temperature (~21oC) for up to several days.
A challenge of large DNA analysis (>50 kb) is to avoid significant DNA breakage
during sample preparation and handling. Sample manipulations must be done
slowly and carefully. Since some amount of shearing is unavoidable, the sample
composition (relative amount of different DNA markers, clones) is important to
achieve good results. If too much T4 or clone DNA is added, the bursts from
sheared fragments mask the smaller marker peaks; if too little T4 DNA or clone
is added, their peak heights would be small with the same data collection time
and there would be more uncertainties in locating the peaks. DNA shearing in
the flow system is relatively insignificant: flowing samples 3-fold faster did not decrease the relative peak areas of larger fragments
in the data.
Pulsed field gel electrophoresis (PFGE)
PFGE was carried out on a CHEF-DR II (Bio-Rad Laboratories, Richmond, CA). Conditions were the following: 1%
GTG Seaplaque agarose (FMC BioProducts, Rockland, ME), 0.5* TBE (45 mM Tris-borate, 1 mM EDTA, pH 8) running buffer; 16oC buffer temperature; switch times ramped from 2.0 to 30 s;
200 V; 15 h running time. The current was typically between 0.17 and 0.22 A.
Gels were stained with ethidium bromide and visualized under UV light.
Ultrasensitive flow cytometry (FCM)
The experimental apparatus is similar to that described previously (
10
,
12
). The excitation light, provided by a cw Ar
+
/Kr
+
laser operated at 514.5 nm, was coupled into a single mode optical fiber. The
output from the fiber was collimated and focused onto a sheath flow cuvette
using a spherical lens with a 25 cm focal length. The 1/e
2
beam diameter at the center of the flow cuvette was measured to be ~46 [mu]m by translating a razor blade through the beam. The excitation laser
power in the flow cell was measured to be 20-30 mW. The fused silica square bore cuvette has a (250 [mu]m)
2
internal dimension and ~100 [mu]m detection side thickness. Fluorescence was collected at 90o to both the optical and flow axes with a 40*, NA 0.85 microscope objective. The collected light was
focused onto a 1.2 (horizontal) * 3.0 (vertical) mm slit (spatial filter) located at the image plane of
the objective, passed through a 550 +- 15 nm interference filter, and focused onto the photocathode of a
thermoelectrically cooled (-30oC) PMT (RCA, Model 31034A, Sommerville, NJ). The detection volume of
~10 pl was defined by the image of the spatial filter in the flow cell, the
focused laser beam and the sample flow stream. Photoelectron pulses were
amplified, discriminated, and counted on a multichannel scaler (MCS). The MCS
summed the number of pulses in 40.96 [mu]s bins. Two hundred scans of data with 16384 bins per scan were collected
and transferred to a Macintosh computer and analyzed by a program written in
the LabVIEW language (National Instruments, Austin, TX). The total data
collection time was 134 s.
Sample solution and sheath fluid were introduced into flow cell by gravity feed.
The sample and sheath flow rates were controlled by height differences between
the sample tube, the sheath bottle and the drain container. Ultrapure water
(Millipore, Bedford, MA) was used as the sheath fluid. Transit times of the
stained fragments through the sample volume were controlled by the sheath flow
rates (~30 [mu]l/min, corresponds to a linear velocity of 2-4 cm/s at the center of the cuvette and a corresponding transit
time through the laser beam of 1-2 ms). The sample flowed at a rate of ~0.2 [mu]l/min, or ~40 fragments/s. The probability of two fragments being in
the sample volume at the same time is given by Poisson statistics as p(2) = 1 - exp(-[tau] * r), where [tau] is the transit time and r is the count rate (
23
). Under our conditions, the probability of double occupancy is ~5%. The point at which double occupancy becomes a problem depends on the
application and can be calculated from the above formula.
An important factor to achieve good resolution in FCM is to maintain a narrow
and stable sample stream. To achieve this, the flow system has to be free of
contaminants and air bubbles. Periodically, the flow system was disassembled
and parts were sonicated in 2% RBS (Pierce, Rockford, IL) methanol/H
2
O (1:1) solution and rinsed with ultrapure water. Before running samples, air
bubbles were removed by flowing methanol, followed by degassed methanol/H
2
O (1:1) through the flow system. Microspheres (Yellow-green Fluoresbrite, diameter 0.997 +- 0.026 [mu]m, Cat# 18860, Polysciences Inc., Warrington, PA) were used to
align the optics to maximize the signal as monitored by an oscilloscope.
Data analysis
Transit times of 1-2 ms were determined by autocorrelation of the first scan of raw data (
12
). The background was determined by averaging the data below a level set near
the maximum of the background noise. The whole data set was scanned for bursts.
A burst was recorded when a series of points exceeded a threshold set above the
average background. A typical background rate for the data discussed below was
5 photoelectrons per MCS bin (40.96 [mu]s), and a typical threshold was chosen to be 6 photoelectrons per bin. The
criterion for choosing the proper threshold was discussed in detail (
12
). For large DNA fragments, the value chosen for the threshold is not critical.
The areas of the bursts were integrated, and histogramed to give a burst size
distribution using Sigmaplot software (Jandel Scientific, San Rafael, CA).
Histograms were fit to a sum of Gaussians plus an exponentially decaying
background (Figs
1
-
4
and Fig.
6
). Burst size means (centroids of histogram peaks) of the DNA standards
determined by the fit were plotted versus the fragment lengths and were fit by
linear regression. The unknown DNA sizes were determined by their burst size
means and the linear regression function.
Optical saturation measurements
Detector saturation represents one potential problem when measuring the photon
bursts of large DNA fragments. To characterize the saturation limit of our PMT,
histograms of burst sizes were obtained from yellow-green Fluoresbrite beads with different neutral density filters (OD = 0-2.0) placed in the detection path to attenuate the fluorescence.
The log of burst size means (pe/ms) were plotted versus OD (a linear plot is
expected if the detector is not saturating). We found that our detector did not
saturate when the burst size mean was below 4000 pe/ms. The largest DNA we
sized (T4 DNA) has a burst size mean of ~2700 pe/ms, well below the saturation point.
RESULTS
Sizing of PAC clone Cl-29 by FCM
Figure
1
a shows the histogram of a sample containing a mixture of TOTO-1 stained [lambda]
Kpn
I digest (1.5, 17.1 and 29.9 kb), [lambda] DNA (48.5 kb), T4 DNA (167 kb) and supercoiled PAC clone Cl-29. [lambda]
Kpn
I digest, [lambda] DNA and T4 DNA were used as size standards. The histogram is the result
of analyzing 134 s of data obtained from ~5000 DNA fragments (this is <1 pg of DNA). Five fragments were resolved while, in this data, the
signature of the 1.5 kb fragment was masked by the background due to residual
scattered light, fluorescent impurities, and debris due to shearing of the
large fragments. The histogram was fit to a sum of 5 Gaussians: [A
i
exp{-[(x -
x
i
)/[sigma]
i
]
2
/2}, where A
i
is the amplitude of a given peak,
x
i
is the burst size mean, [sigma]
i
is the standard deviation, x is the burst size, and i = 1-5] and a decaying exponential [[alpha] exp(-[beta]x), where [alpha] and [beta] are constants] with A
i
,
x
i
, [sigma]
i
, [alpha] and [beta] as fitting parameters. The results are summarized in Table
1
and the resulting curve is shown in Figure
1
a. The burst size means from the Gaussian fits were plotted versus the lengths
of the DNA standards (17.1, 29.9, 48.5, 167 kb) and were fit by a linear
regression (Fig.
1
b). The linear correlation coefficient is 0.99997; the slope of the resulting
line is 27.0 +- 0.2 pe/kb and the intercept is -28 +- 14 pe. The uncertainties are the standard deviations of
the resulting parameters as reported by Sigmaplot. Deviations of the measured
values from the fitted line are also listed in Table
1
. The average absolute deviation is 1.7%. The unknown size of Cl-29 was calculated using the linear regression function and the burst size
mean of Cl-29 from the Gaussian fit as listed in Table
1
. The obtained Cl-29 size is 88.9 +- 0.8 kb. Here, the standard deviation of the clone size was
calculated using error propagation theory.
.
Parameters obtained from the histograms of sizing PAC clone Cl-29 (Figs 1 and 2)
Fragment
Amplitude
Mean
Standard
CV(%)
Shot noise(%)
Deviations from
length (kb)
(A
i
)
(
x
i
)
deviation ([sigma]
i
)
([sigma]
i
/
x
i
)
([radic]
x
i
/
x
i
)
linear fit (%)
a
17.1
35.3 +- 1.3
b
453.4 +- 1.3
b
29.8 +- 1.3
b
6.6
4.7
-4.5
Figure 1
29.9
26.7 +- 1.2
771.7 +- 1.9
38.1 +- 1.9
4.9
3.6
1.0
Sizing intact
48.5
30.2 +- 1.0
1266.7 +- 2.0
54.5 +- 2.0
4.3
2.8
1.2
Cl-29
88.9
23.3 +- 0.7
2372.3 +- 3.7
108.2 +- 3.8
4.5
2.1
167
28.3 +- 0.6
4486.9 +- 3.2
122.1 +- 3.3
2.7
1.5
-0.1
Figure 2
16.2
14.6 +- 0.8
358.4 +- 2.1
32.8 +- 2.2
9.1
5.3
Sizing Cl-29
48.5
17.1 +- 0.7
1066.8 +- 2.3
50.3 +- 2.3
4.7
3.1
insert and
73.7
9.7 +- 0.6
1617.9 +- 4.5
63.5 +- 4.6
3.9
2.5
vector
167
13.3 +- 0.5
3663.2 +- 4.3
109.2 +- 4.4
3.0
1.6
a
The deviation of the data from the linear regression fit is defined as: (fitted
burst size - measured burst size)/fitted burst size.
b
The uncertainties are the standard deviations of the resulting parameters as
reported by Sigmaplot.
.
Parameters obtained from the histograms of sizing PAC clone Cl-26 (Figs 3, 4)
Fragment length
Amplitude
Mean
Standard
(kb)
(A
i
)
(
x
i
)
deviation ([sigma]
i
)
17.1
76.1 +- 2.2
328.3 +- 0.8
23.4 +- 0.8
Figure 3
29.9
38.8 +- 1.8
540.3 +- 1.8
34.4 +- 1.9
Sizing intact
48.5
41.7 +- 1.5
871.5 +- 2.1
52.4 +- 2.2
Cl-26
119.1
18.0 +- 1.1
2129.4 +- 6.7
99.3 +- 7.1
167
11.8 +- 0.9
2981.8 +- 12.4
145.1 +- 13.0
Figure 4
15.5
35.4 +- 2.1
236.6 +- 1.9
28.9 +- 2.1
Sizing Cl-26
48.5
103.4 +- 1.6
764.6 +- 0.9
50.0 +- 0.9
insert and
105.3
15.3 +- 1.1
1673.6 +- 8.2
98.0 +- 8.7
vector
167
17.8 +- 0.9
2658.4 +- 8.7
151.7 +- 9.4
Supercoiled PAC clone Cl-29 was digested with
Eag
I. This enzyme cuts at the vector and insert junction sites (
18
). The released linear restriction fragments were sized by FCM with [lambda] DNA and T4 DNA as size standards and the results are shown in Figure
2
a. The histogram was fit to a sum of 4 Gaussians plus a decaying exponential.
The resulting parameters A
i
,
x
i
and [sigma]
i
(i = 1-4) are summarized in Table
1
and the resulting curve is shown in Figure
2
a. The burst size means from the Gaussian fits were plotted versus DNA fragment
lengths, and a line was drawn through the points of DNA standards of 48.5 and
167 kb (Fig.
2
b). The slope of the resulting line is 21.9 +- 0.1 pe/kb and the intercept is 4 +- 4 pe. The unknown insert and vector sizes were calculated using
the function of the resulting line and the burst size means from the Gaussian
fits as listed in Table
1
. The obtained Cl-29 insert size: 73.7 +- 0.4 kb; Cl-29 vector size: 16.2 +- 0.2 kb. The sum of the Cl-29 insert and its vector, 89.9 +- 0.5 kb, agrees within experimental
error with the size obtained for the intact supercoiled Cl-29 (88.9 +- 0.8 kb, Fig.
1
). The size of the PAC vector (pCYPAC2) was treated as unknown here. A closely related PAC vector (pCYPAC1) has a reported size of ~17 kb (
18
).
Sizing of PAC clone Cl-26 by FCM
The same approach was used to determine the sizes of supercoiled PAC clone Cl-26 and its linear restriction fragments by
Eag
I. Figure
3
a shows the histogram of a sample of TOTO-1 stained supercoiled Cl-26 with [lambda]
Kpn
I digest, [lambda] DNA and T4 DNA as size standards. Figure
3
b shows the plot of the burst size means from the Gaussian fits versus the DNA
fragment lengths. The Gaussian fit parameters A
i
,
x
i
and [sigma]
i
(i = 1-5) are summarized in Table
2
and the fitting curve is shown in Figure
3
a. The linear correlation coefficient of Figure
3
b is 0.99998; the slope of the linear regression line is 17.8 +- 0.1 pe/kb and the intercept is 15 +- 7 pe. The obtained supercoiled Cl-26 size is 119.1 +- 0.9 kb.
Sizing of supercoiled and linear PAC clones by PFGE
To check the validity of PAC clone sizing by FCM, the intact supercoiled clones
isolated from
E.coli
host cells and their linear restriction fragments were also sized by PFGE.
Approximately 0.2 [mu]g of clone DNA was loaded into each gel well. The pulsed-field gel was run for 15 h with switch times ramped from 2.0 to 30 s.
Figure
5
shows a photograph of the ethidium bromide stained gel. Lanes 1, 6, 7, 16 are
Low Range PFG markers (sizes indicated on the figure); lanes 2 and 8 are intact
supercoiled Cl-26 from two different preps; lanes 3 and 9 are
Eag
I restriction fragments of intact supercoiled Cl-26, showing two bands, corresponding to the linear insert and vector;
lanes 4 and 10 are intact supercoiled Cl-29 from two different preps; lanes 5 and 11 are
Eag
I restriction fragments of intact supercoiled Cl-29; lanes 12-15 are not relevant. To estimate the sizes of DNA fragments, the
migration distances of all the bands from the loading wells were measured using
a ruler and the lengths of markers were plotted versus their corresponding
migration distances. The resulting plot was fit to a 5th order polynomial
function and the sizes of the clones were estimated using their migration
distances and the polynomial function obtained from the fit to the markers. The
estimated linear Cl-29 insert size is 71.0 +- 7.1 kb, where the uncertainty represents a typical 10% error
quoted for PFGE (
6
). The obtained Cl-29 insert size is in good agreement with that measured by FCM (73.7 +- 0.4 kb, Fig.
2
). The estimated linear Cl-26 insert size from the gel is 107.1 +- 10.7 kb, which is also in good agreement with that measured by
FCM (105.3 +- 0.9 kb, Fig.
4
). The estimated size of the linear vector is 16.7 +- 1.7 kb, also agrees with that determined by FCM (16.2 +- 0.2 kb, Fig.
2
; 15.5 +- 0.3 kb, Fig.
4
). The intact supercoiled Cl-26 and Cl-29 clones did not migrate appreciably, which is not surprising since
large supercoiled DNA migrates anomalously in gel electrophoresis (
5
,
24
). Sizing of PAC clones by PFGE requires enzyme digestion to linearize
supercoiled DNA.
Sizing of PAC clones directly from the PAC clone library by FCM
Figure
6
shows four representative histograms of sizing clones directly from the PAC
clone library. Here [lambda]
Kpn
I digest, [lambda] DNA and T4 DNA were used as markers (same as Figs
1
and
3
). The data analysis was the same as that employed to analyze data for intact
supercoiled Cl-29 and Cl-26. The obtained sizes for supercoiled Cl-7, Cl-10, Cl-32 and Cl-56 are listed in Table
3
. The insert sizes, calculated by subtracting the vector size (15.9 +- 0.4 kb, Figs
2
and
4
), are in agreement with those obtained by PFGE within experimental uncertainty
(Table
3
). The apparent systematic discrepancy derived from these two methods is not
significant: in general, both positive and negative deviations are observed
(see above section).
.
Comparison of the sizes of clones from the PAC library measured by FCM and PFGE
Clone
By FCM (insert + vector, kb)
By FCM (insert only, kb)
By PFGE (insert only, kb)
Cl-7
105.0 +- 1.7
89.1 +- 1.7
97 +- 10
Cl-10
101.7 +- 1.8
85.8 +- 1.8
93 +- 9
Cl-32
95.5 +- 0.8
79.6 +- 0.9
81 +- 8
Cl-56
103.2 +- 1.9
87.3 +- 1.9
94 +- 9
Estimating DNA concentration based on the histogram peak areas
DISCUSSION
Accuracy
Sizing of large DNA fragments by FCM is more accurate (~2% uncertainty) than by PFGE, which is generally considered to have a 10% uncertainty in estimating sizes (
6
). This conclusion is based upon the following: (i) the average absolute deviation from the linear fits for
large DNA standards is 1.7% [Fig.
1
; the 17.1 kb point, which has the largest deviation (-4.5%), is always above the line as observed previously (
10
,
12
)]; (ii) the results are reproducible from run-to-run and day-to-day: the average precision of sizing the same PAC clone is 1.7%; and (iii) the signal in FCM
(photon burst) is linear with fragment length, while the signal in PFGE (migration distance) is non-linear with DNA length.
Resolution
When the instrument is optimized, photon shot noise ([radic]
x
i
/
x
i
) accounts for half or more of the observed coefficient of variation (CV)
(Table
1
). Larger fragments have relatively better resolution since the shot noise is
relatively smaller due to their larger burst sizes. For a given fragment, a
larger burst size will result in a better CV. Larger burst sizes could be
attained by increasing the laser power or using brighter dyes. The signal count
rate (pe/ms) achievable is ultimately limited by detector saturation (see
Materials and Methods section). Larger burst sizes could be obtained without
saturation by increasing the transit time. Our resolution on large fragments (2-5% for >50 kb DNA) is comparable with or better than that of PFGE (
25
).
Sensitivity
Sensitivity is an important issue in characterizing clones obtained in minute
quantities (such as single copy PAC and BAC clones). As little as 0.4 pg of
total DNA (or ~0.1 pg of clone DNA) is analyzed to generate a histogram that allows
accurate sizing of clones. Although the staining solution was made up at ~10
-11
M in the currently reported experiments, good results were obtained in other
work for sizing a mixture of [lambda] DNA and [lambda]
Kpn
I digest stained directly at ~10
-13
M with TOTO-1. Only ~2 ng of clone DNA are needed to make up 1 ml of 10
-13
M working solution, of which only ~0.5 [mu]l is analyzed to generate one histogram. Improvements in sample
handling will reduce further the volume of solution needed for analysis. The
amount of DNA loaded per gel well in Figure
5
is ~0.2 [mu]g. Thus, sizing of DNA by FCM requires orders of magnitude less DNA
than does PFGE.
Signal linear with the number of fragments
FCM analyzes and classifies DNA fragments one at a time. The relative numbers
counted for different DNA fragments correspond to the molar concentrations of
the fragments in the sample solution. Unknown DNA concentrations can be
estimated with >80% accuracy based on the analysis of their relative peak areas
in the histogram.
Effect of DNA conformation on sizing
The sizes of intact supercoiled Cl-29 and Cl-26 obtained by FCM are in good agreement with the sums of the sizes
of their linear restriction fragments (see Results section). These results demonstrate that FCM can be applied to size both linear and supercoiled clones. Two implications can be drawn from these results: (i) DNA conformation (supercoiled or linear) does not affect the stoichiometric binding of TOTO-1 to DNA double strands; and (ii) DNA conformation does not affect the
linear relationship between photon burst size and the number of dye molecules
associated with the DNA strands. In contrast, large supercoiled DNA migrates
anomalously in PFGE and it is essential to digest enzymatically the supercoiled
clones into linear molecules before they can be sized accurately.
CONCLUSIONS
We have demonstrated a flow cytometry-based technique to size large DNA fragments and its application to the
characterization of P1 artificial chromosome clones. This technique is superior
to the current most commonly used technique (PFGE) for the following reasons:
(i) the data are acquired rapidly (<3 min compared with 15 h for PFGE); (ii) the technique is sensitive (<1 pg of DNA is analyzed compared with 0.2 [mu]g of DNA for PFGE); (iii) the measurement is linear with both fragment
length and number of fragments; and (iv) the results are conformation
independent as both linear and supercoiled PAC clones were sized accurately. We
anticipate that this method will play an important role in characterizing
PAC/BAC clone libraries that are widely used in gene mapping, sequencing, and
other genetic analyses. The primary challenge of sizing large DNA in solution
is to avoid significant DNA breakage during sample preparation and handling.
Since DNA as large as 1 * 10
6
bp has been handled successfully in the genome community (with YAC cloning
systems), we anticipate our DNA sizing range can be pushed well beyond 167 kb.
This method also holds the potential for scale-up through multiplicity and automation.
ACKNOWLEDGEMENTS
This work was supported by internal funding from Los Alamos National Laboratory,
by the DOE funded Los Alamos Center for Human Genome Studies (W-7405-ENG-36), and by the NIH funded National Flow Cytometry Resource
(RR-01315). We thank Harvey Nutter for his valuable technical assistance.
REFERENCES
1 Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Habor, NY, pp. 6.5, pp. 6.37.
23 Lindmo,T., Peters,D.C. and Sweet,R.G. (1990) In Melamed,M.R., Lindmo,T. and Mendelsohn,M.L. (eds), Flow Cytometry and Sorting, 2nd edition. Wiley-Liss, Inc., New York, pp. 159.
