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BACKGROUND OF THE INVENTION
This invention relates generally to the field of nucleic acid hybridization
on membranes. More particularly, this invention relates to a method for
automated multiplex sequencing of DNA.
Large scale nucleotide sequencing initiatives, such as a project to
sequence the human genome, have created a need for increased efficiency
and productivity. J. Watson, 248 Science 44 (1990). Automation of the
various steps involved in sequencing is one area in which gains in
efficiency and productivity are being made.
Multiplex sequencing, one scheme for reducing the number of sequencing
reactions and electrophoresis steps, involves the processing of a mixture
of sequencing templates followed by sequential hybridization with selected
probes. G. Church & S. Kieffer-Higgins, 240 Science 185 (1988); U.S. Pat.
No. 4,942,124. In this method, many sequencing templates, each carrying a
short known sequence or tag, are processed together. A single DNA
preparation yields a mixture of templates. Sequencing reactions are
performed on the mixture in the absence of any label, and the mixed
reaction products are fractionated by electrophoresis, transferred to a
membrane, and probed sequentially by hybridization with labeled
oligonucleotides specific for each tag. Each hybridization step reveals
the nucleotide sequence of one component of the template mixture. Between
hybridizations the labeled probe is removed to permit the next
hybridization without interference from the previous probe. The advantages
of multiplex sequencing come from the parallel processing of template
preparations and sequencing reactions, and the simultaneous
electrophoresis of mixtures of templates. Multiplex sequencing can reduce
the time, effort, and resources needed for these steps by about a factor
of the number of different sequencing templates in the mixture.
The savings made in sequencing reactions and electrophoresis by multiplex
sequencing are offset to some extent, however, by new steps that are
unnecessary in conventional sequencing protocols. Hybridization of the
membrane is an added step that is repeated with each specific probe.
Fortunately, however, the hybridization process is automatable. P.
Richterich et al., 7Bio/Techniques52 (1989). A remaining problem is the
acquisition of sequence data in electronic form. Automated sequencing
machines are available that detect fluorescently labeled sequencing
products as they migrate through a gel. The data acquired in this way are
then interpreted by an algorithm that yields a called sequence. Most
large-scale sequencing efforts have turned toward such machines as the
only way of obtaining sufficient efficiency.
Conventionally, hybridization probes have been labeled with radioisotopes.
Although radioactive probes can detect minute quantities of DNA, they are
hazardous and unstable, and high-resolution direct imaging of radioactive
signals is not straight-forward. Non-radioactive methods of DNA detection
have been developed in recent years. The most sensitive methods involve
enzymatic conversion of substrates to colored, J. Leary et al., 80 Proc.
Nat'l Acad. Sci. USA 4045 (1983), or chemiluminescent products, J. Voyta
et al., 34 J. Clin. Chem. 1157 (1988); A. Schaap et al., 28 Tetrahedron
Lett. 1159 (1987); I. Bronstein et al. 180 Anal. Biochem. 95 (1989). In
this approach, an enzyme is linked to a probe, and an enzyme substrate
that yields a colored or chemiluminescent product is applied to the
membrane. After the enzyme acts on the substrate, the result is a pattern
of color or light corresponding to the pattern of the target DNA on the
membrane. Although colorimetric detection of sequence ladders has been
achieved, P. Richterich et al, 7 Bio/Techniques 52 (1989), the inability
to remove the colored product from the membrane precludes its use for
sequential probing.
As currently practiced, automated DNA sequencing makes use of fluorescent
labels for DNA detection. L. Smith et al., 321 Nature 674 (1986); W.
Ansorge et al., 15 Nucleic Acids Res. 4593 (1987); J. Prober et al., 238
Science 336 (1987). In these methods fluorescence detection occurs while
the DNA is in the gel. Under such conditions, a single fluorescent moiety
per DNA molecule is sufficient for detection. Attempts at fluorescent
detection in multiplex sequencing revealed a grossly inadequate limit of
detection for DNA sequencing purposes. A. Karger et al., 206 Proc. SPIE 78
(1990). Background fluorescence from most membranes adds large quantities
of noise, T. Chu et al., 13 Electrophoresis 105 (1992); U.S. Pat. No.
5,112,736, so that a much more intense signal is required to achieve an
adequate signal-to-noise ratio than is required in a gel. Low fluorescence
membranes, such as amine derivatized polypropylene (e.g., U.S. Pat. No.
5,112,736), are known, however such low flourescence membranes are
restricted by a limit of detection about 100-fold too high for multiplex
sequencing and the membranes are more fragile than nylon membranes.
Chemiluminescent hybridization signals are typically imaged by exposure to
X-ray film although other methods are known, such as with a CCD
(charge-coupled device) camera. U.S. Pat. No. 5,162,654. However the light
output from chemiluminescence is quite low. Although enzymatic turnover
results in many chemiluminescent molecules per target DNA molecule, at
most one photon is emitted for each product molecule produced and in
practice there is only about 1 photon emitted per 10.sup.4 molecules. Due
to the low level of light emitted, a sensitive, low-noise detector, such
as a cryogenically cooled CCD, is required for imaging, and a long
exposure time is needed. A fully automated system based on
chemiluminescence could be constructed, but it would be expensive and
slow.
In the most straightforward operational mode, a CCD image is acquired as a
snapshot, analogous to the operation of a photographic camera. The major
advantages of digital imaging, in particular fast visualization, high
sensitivity, quantitative imaging, and computer readable format, have been
well documented. E. Ribeiro et al., 194 Anal. Biochem. 174 (1991); P.
Jackson et al., 9 Electrophoresis 330 (1988); P. Jackson, 270 Biochem. J.
705 (1990); K. Chan et al., 63 Anal. Chem. 746 (1991); M. Lanan et al., 31
Biopolymers 1095 (1991); M. Lanan et al., 64 Anal. Chem. 1967 (1992); A.
Karger et al., 1206 Proc. SPIE 78 (1990); K. Misiura et al. 18 Nucleic
Acids Res. 4345 (1990); D. Pollard-Knight et al., 185 Anal. Biochem. 84
(1990); Z. Boniszewski et al., 11 Electrophoresis 432 (1990). When
compared to other methods of visualization, however, such as
autoradiography using isotope labels and X-ray film, the most obvious
limitation of CCD imaging lies in the dimensions of the sensor arrays most
commonly used in analytical applications. Their limited size rules out the
recording of high-resolution electropherograms on a single frame. The
large number of bands that can be resolved by high-resolution
electrophoretic methods far exceeds the number of bands that can be
adequately sampled on arrays having 512 to 768 CCD elements along their
long axis, such as those referenced above.
One solution to obtaining a CCD image with adequate sampling over the
entire surface of sequencing electropherograms is by manually merging
partially overlapping individual frames on a computer screen using an
image analysis tool. P. Jackson, 270 Biochem. J. 705 (1990). However, this
procedure is time consuming and labor intensive, and the quality of the
resulting composite image is compromised by discontinuities.
Another solution would be to use larger CCD arrays. CCD arrays consisting
of 2048 elements square are commercially available, although at prices
that are often prohibitive for analytical applications. Considering that
several thousand data points need to be collected when several hundred
bands are being separated, even a state-of-the-art, 4-megapixel CCD area
array will fall short of the most demanding requirements of
high-resolution separations. DNA sequencing, for example, requires
sampling capability for well above 500 bands on a single lane, translating
into much more than 2048 data points.
Continuous data acquisition using an area CCD can be achieved by operating
the CCD camera in Time Delay and Integration (TDI) mode. Line scan CCD
cameras are also available, but TDI mode provides greater sensitivity than
line scan. TDI operation adds the capability of continuous data
acquisition independent of the array length. This has been shown for two
high-speed, fluorescence DNA sequencing formats: capillary
electrophoresis, A. Karger, et al., 18 Nucleic Acids Res. 4955 (1991), and
ultrathin slab gels, A. Kostichka et al., 10 Bio/Technology 78 (1992). TDI
mode has also been used to monitor migrating fluorescent bands in
capillary electrophoresis along the length of the column with a CCD
camera. J. Sweedler et al., 63 Anal. Chem. 496 (1991). A TDI system for
fluorescence detection on membranes is needed for automation of multiplex
sequencing.
The task of converting relative band positions into nucleotide sequence is
conceptually simple, however, the 1-3% error rate of human readers
indicates that reading is more complex in practice. Band amplitudes and
positions vary due to enzyme behavior and other biochemical factors, and
instrumentation and handling factors, such as uneven temperature
distribution. Band positions as a function of fragment size typically
follow either quasi-logarithmic or constant spacing rules, depending on
the instrumentation, but spatial jitter and position anomalies can be
large enough to superimpose adjacent bands. Interlane band amplitudes
vary, and intralane band amplitudes change both locally and along the
length of the lane. Across a given electrophoretic gel, bands change width
and may be tilted or take on complex shapes. Automated sequence readers
must be able to deal with all this variation.
OBJECTS AND SUMMARY OF THE INVENTION
It is an object of the present invention to provide a system and method for
automated multiplex sequencing of DNA.
It is also an object of the invention to provide an apparatus for automatic
hybridization and washing of sequencing membranes, detection and imaging
of fluorescent signals, and producing a called sequence with existing
software from sequence data on the sequencing membranes.
An additional object is to provide an apparatus capable of handling other
membrane-based detection methods such as colony and plaque hybridizations;
Southern, Northern, and Western blot procedures; multiplex genotyping of
simple sequence repeats; sequencing and mapping by hybridization; and dot,
slot, and allele-specific oligonucleotide blot techniques.
It is another object of the invention to provide a system and method for
detecting DNA on sequencing membranes that is compatible with automated
multiplex sequencing.
It is a further object of the invention to provide a system and method for
imaging fluorescent signals on sequencing membranes that is compatible
with automated multiplex sequencing and which is also applicable to other
membrane-based detection methods.
Yet a different object is to provide a method for enzyme-linked fluorescent
detection of membrane-bound nucleic acid.
These and other objects may be accomplished by a method for sequencing a
nucleic acid specimen by automated multiplex sequencing comprising the
steps of (a) preparing multiplex sequencing reaction products, (b)
separating the sequencing products according to their size, (c) attaching
the separated sequencing products to a membrane, (d) placing the membrane
in a chamber device of an integrated automated imaging hybridization
chamber system comprising an hybridization chamber device, means for fluid
delivery to the chamber device, imaging means for light delivery to the
membrane and image recording of fluorescence emanating from the membrane
while in the chamber device, and controller means for controlling the
operation of the system, (e) introducing a first oligonucleotide probe
containing an enzyme binding moiety, the probe capable of specifically
hybridizing with a tag sequence on the membrane-bound sequencing products,
into the chamber device by the fluid delivery means and thereby
hybridizing the probe to the fractionated products, (f) introducing an
enzyme into the chamber device by the fluid delivery means and binding
enzyme to the binding moiety on the first oligonucleotide probe, (g)
introducing a fluorogenic substrate into the chamber device by the fluid
delivery means and contacting the enzyme with the substrate so that the
substrate is converted into a fluorescent product, (h) illuminating the
fluorescent product in the chamber device with a beam of light from the
imaging means to excite fluorescence of the fluorescent product and
produce a pattern of hybridization that reflects the nucleotide sequence
of the nucleic acid specimen, (i) imaging the hybridization pattern by the
imaging means and storing the pattern of hybridization as digital signals,
and (j) converting the digital signals by the controller means into a
linear string of nucleotides corresponding to the nucleotide sequence of
the nucleic acid specimen.
Another aspect of the invention is adding the additional steps of (k)
removing the fluorescent product from the membrane by introducing an
appropriate wash solution into the chamber compartment by the fluid
delivery means, (1) introducing a second oligonucleotide probe, containing
a binding moiety to which an enzyme may be bound and which is able to
hybridize specifically with a tag sequence different than the tag sequence
of step (e), into the chamber device by the fluid delivery means and
hybridizing the second probe to the fractionated products, introducing an
enzyme into said chamber device by the fluid delivery means and binding
the enzyme to the binding moiety on the second oligonucleotide probe, and
repeating steps (g) through (j).
Another aspect of the invention is providing a fluorogenic substrate that
is converted into a fluorescent product by an enzyme, wherein enzyme
turnover produces many copies of the fluorescent product, the fluorescent
product produces a clear pattern of hybridization with the support-bound
nucleic acid, and the fluorescent product is easily removed for subsequent
rounds of hybridization. Illustrative of suitable fluorogenic substrates
is the benzothiazole derivative,
2'-(2-benzothiazolyl)-6'-hydroxybenzothiazole phosphate (BBTP). Alkaline
phosphatase catalyzes the conversion of BBTP into the fluorescent product
BBT. BBT does not diffuse on nylon membranes thus providing a sharp
fluorescent image of membrane-bound DNA when illuminated with a wavelength
of light that excites fluorescence. BBT is easily removed from the
membrane by washing in detergent so that subsequent hybridizations can be
performed on the same membrane.
Another aspect of the invention is an automated imaging hybridization
chamber for automatic hybridization and imaging of multiplex sequencing
membranes. The automated imaging hybridization chamber comprises a pair of
concentric nested horizontal cylinders, i.e., a sealed inner cylinder that
is rotatable about an axis and a sealable stationary outer cylinder having
a light transparent window and entry means through which the inner
cylinder may be accessed. The outer surface of the inner cylinder,
containing means for attaching the sequencing membrane thereto, is spaced
apart from the inner surface of the outer cylinder thereby forming an
enclosed chamber compartment, the lower portion of which may be employed
for receiving fluids (referred to as a "fluid puddle") used in the
hybridization process. The portion of the chamber compartment above the
fluid is an air space through which the membrane may be visualized and
subjected to light through the transparent window in the outer cylinder.
The inner cylinder is rotatable on an axle and is coupled to a stepper
motor through gears and a toothed belt for causing the inner cylinder to
rotate. The underside of the outer cylinder contains a plurality of valved
ports extending through the cylinder wall through which fluids may be
injected and removed from the chamber without cross contamination of the
fluids. Each port is either coupled to a fluid delivery module for storing
and delivering the fluids to the chamber compartment or to drain means for
removing fluid from the chamber compartment. One type of fluid delivery
module includes a large capacity reservoir and a metering pump.
Optionally, a batch heater is included for heating the fluids before they
are inserted into the chamber. A second type of fluid delivery module
includes a probe reservoir, a large capacity reservoir, and a dual headed
peristaltic pump for mixing probes with other solutions and delivering the
fluid mixture to the chamber. Probe reservoirs can also be of the
non-mixing type. Optionally, a refrigeration unit may be included to chill
probe or other solutions.
Another aspect of the invention includes an imaging apparatus for imaging
the pattern of hybridization on the membrane while the membrane is within
the chamber. The outer cylinder advantageously includes a window of
optically transparent material through which visible light may pass. A
light source appropriately positioned outside the outer cylinder relative
to the transparent window illuminates the membrane at a wavelength of
light for exciting fluorescence of the fluorescent product on the
membrane. A lens can be used to widen the distribution of the beam of
light. A filter removes unwanted wavelengths of light. A CCD camera also
appropriately positioned outside the outer cylinder relative to the
transparent window operating in Time Delay and Integration mode records an
image of the pattern of hybridization produced by the fluorescence. A
filter removes interfering wavelengths of light from background sources.
To generate an image, the membrane, which is mounted on the inner chamber,
is moved relative to the CCD camera. This movement is caused by rotation
of the inner chamber by the stepper motor and is synchronized with CCD
pixel shifts in the camera. The image is stored electronically.
The above provides an overall summary of a preferred embodiment to a
complete and integrated system and method for multiplex DNA sequencing.
However, portions of the integrated system and/or method taken separately
or combined may be utilized in other embodiments. For example,
hybridization means other than the automated chamber may be utilized in a
multiplexing operation. Also, membrane bound nucleic acids may be detected
by enzyme-linked fluorescence without using the specific hybridization
chamber or the specific imaging means disclosed. Further, the disclosed
hybridization chamber can be used without the disclosed imaging means
and/or fluorescence system.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows a portion of a sequencing membrane corresponding to
nucleotides 290-373 from the sequencing primer at 65 minutes, 110 minutes,
and 20 hours after addition of the fluorogenic substrate MUFP.
FIG. 1B shows a portion of a sequencing membrane corresponding to
nucleotides 290-373 from the sequencing primer at 40 minutes, 110 minutes,
and 23 hours after addition of the fluorogenic substrate 5MFP.
FIG. 1C shows a portion of a sequencing membrane corresponding to
nucleotides 290-373 from the sequencing primer at 40 minutes, 110 minutes,
and 20 hours after addition of the fluorogenic substrate BBTP.
FIG. 2 is a schematic perspective view of the automated imaging
hybridization chamber system according to the present invention.
FIG. 3 shows an enlarged cross sectional view of an illustrative check
valve contained in the automated imaging hybridization chamber shown in
FIG. 2.
FIG. 4 shows an end sectional view of a portion of the automated imaging
hybridization chamber shown in FIG. 2 taken along lines 4--4 of FIG. 2.
FIG. 5 shows a plot of unprocessed one-dimensional traces of a portion of a
sequencing membrane read according to the present invention. The line
patterns represent the four deoxynucleotides: T (solid), C (dashed), G
(dotted), and A (dash-dot).
FIG. 6 shows the detection limit of a membrane-bound 75-mer oligonucleotide
in a single direct transfer electrophoresis sequence band wherein the
75-mer oligonucleotide was probed with a complementary 25-mer
oligonucleotide labeled with a single 5' biotin and detected with
streptavidin-alkaline phosphatase and BBTP.
FIG. 7 shows the detection limit of a membrane-bound 75-mer oligonucleotide
in a single direct transfer electrophoresis sequence band wherein the
75-mer oligonucleotide was labeled directly with a single 5' biotin and
detected with streptavidinalkaline phosphatase and BBTP.
DETAILED DESCRIPTION OF THE INVENTION
Before the present method of automated multiplex nucleotide sequencing is
disclosed and described, it is to be understood that this invention is not
limited to the particular process steps and materials disclosed herein as
such process steps and materials may vary somewhat. It is also to be
understood that the terminology used herein is used for the purpose of
describing particular embodiments only and is not intended to be limiting
since the scope of the present invention will be limited only by the
appended claims and their equivalents.
As used herein, "membrane" includes thin films composed of nylon,
nitrocellulose, polypropylene, and the like, as well as their functional
equivalents now known in the art or later developed. Also, other solid
supports to which a nucleic acid may be bound, hybridized and detected by
enzyme-linked fluorescence is considered a functional membrane equivalent
even if not in the form of a thin film.
As used herein, "macromolecule" means a nucleic acid or protein or their
functional equivalents. For example, nucleic acid is intended to include
naturally occurring and synthetic oligonucleotides and polynucleotides
regardless of whether they contain ribose, deoxyribose, or dideoxyribose
sugars or a combination thereof, and regardless of whether they are single
stranded, double stranded, or a combination thereof. Protein is intended
to include oligopeptides and polypeptides, whether naturally occurring or
synthetic.
Enzyme-Linked Fluorescent Detection of DNA
One way of increasing fluorescent light output is to make use of enzymatic
turnover to yield many fluorescent molecules per target DNA molecule.
Fluorogenic substrates, compounds exhibiting low fluorescence which yield
highly fluorescent products when acted upon by an enzyme, are available
for several enzymes. Each molecule of enzyme can catalyze the production
of many fluorescent molecules. The following fluorogenic substrates for
calf intestinal alkaline phosphatase were tested as agents for
visualization of probed DNA sequence ladders on nylon membranes:
.beta.-methylumbelliferyl phosphate (MUFP), 5-methyl fluorescein phosphate
(5MFP), and 2'-(2-benzothiazolyl)-6'-hydroxybenzothiazole phosphate
(BBTP). MUFP is commonly used in fluorescent assays for alkaline
phosphatase, whereas 5MFP is a weakly fluorescent compound that, when
hydrolyzed, yields the methyl ether of the common fluorophore,
fluorescein. BBTP, also known as "ATTOPHOS," R. Cano et al., 12
Bio/Techniques 264 (1992), is hydrolyzed by alkaline phosphatase to the
highly fluorescent 2'- (2-benzothiazolyl) -6'-hydroxybenzothiazole (BBT).
R. Klem & W. Marvin, Preparation and Use of Fluorescent Benzothiazole
Derivatives, PCT WO 90/00618 (Jan. 25, 1990). While BBTD is the preferred
substrate thus far considered, the invention is not limited to any
particular chemical structure for a fluoregenic substrate. Any substrate
which does not diffuse on the membrane of choice thereby providing a sharp
fluorescent image of membrane-bound DNA when illuminated with a wavelength
of light that excites fluorescence is suitable. Preferably the substrate
will also be is easily removed from the membrane by washing in detergent
so that subsequent hybridizations can be performed on the same membrane.
Example 1
A sequencing reaction was prepared using the dideoxynucleotide chain
termination method of F. Sanger et al., 74 Proc. Nat'l Acad. Sci USA 5464
(1977), with 1 pmol of M13mp19 template DNA and T7 DNA polymerase.
Single-stranded template DNA was prepared from M13mp19 phage grown and
purified from E. coli 71-18 using polyethylene glycol/NaCl precipitation
followed by phenol/chloroform extraction and ethanol precipitation. J.
Messing et al., 9 Nucleic Acids Res. 309 (1981). For sequencing, 2 pmol of
"universal" sequencing primer and 1 pmol of template DNA were annealed in
a 10 .mu.l volume containing 4 mM MnCl.sub.2, 80 mM Tris. HCl, pH 7.6, 30
mM sodium isocitrate, 10 mM dithiothreitol, and 0.1 .mu.M of each of the
four deoxynucleoside triphosphates. Annealing was at 65.degree. C. for two
minutes, then at 37.degree. C. for 15 minutes, before addition of 2 units
of T7 DNA polymerase (Sequenase 2.0, U.S. Biochemicals). After 1 minute at
room temperature, 2.5 .mu.l of this mixture was placed into each of four
microtiter wells containing 2.5 .mu.l of the appropriate deoxy(dNTP) and
dideoxy-(ddNTP) nucleotides. All d/ddNTP mixtures contained 300 .mu.M dNTP
and 1 .mu.M of the appropriate ddNTP. The reaction was incubated at
37.degree. C. for 8 minutes and terminated by the addition of 6 .mu.l of
stop solution containing 98% formamide, 0.1% xylene cyanol, and 0.1%
bromphenol blue, and heating on a boiling water bath for 3 minutes.
Reaction products were loaded in three sets of four lanes each
(corresponding to the four deoxynucleotides A, C, G, and T), and were
fractionated and then transferred to a nylon membrane. A 4% acrylamide
slab gel (Long Ranger Gel Solution, AT BioChem Inc., Malvern, Pa.)
containing 1.times.TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM
EDTA, pH 8.3) was poured between 80 cm.times.35 cm glass plates using a
0.2 mm to 0.1 mm reverse wedge spacer. Gels were prerun at 50 V/cm, then
0.75 .mu.l of sequence reaction was loaded per lane. DNA fragments were
fractionated at 50 V/cm with passive thermostating provided by aluminum
plates clamped to both glass surfaces. The electrophoresis system was a
direct transfer apparatus, F. Pohl & S. Beck, 155 Meth. Enzymol. 250 (R.
Wu ed., 1987) and hereby incorporated by reference, containing a custom
stepper motor and controls, that pulls the membrane at a constant rate
perpendicular to the bottom edge of the glass plates, thus, transferring
the DNA fragments to the membrane. Conventional methods of transferring
DNA from gels to membranes, such as electroblotting as described in U.S.
Pat. No. 5,112,736, would also be adequate. After electrophoresis and
transfer, the membrane was removed from the apparatus, rehydrated in 40 mM
sodium phosphate buffer, pH 7.6, and irradiated with a total of 225
mJ/cm.sup.2 of UV light to cross link the DNA fragments to the nylon
membrane. Unbound DNA was removed by washing the membrane in PBS (40 mM
sodium phosphate, pH 7.6, 150 mM NaCl), 5% SDS, and then the membranes
were stored in the same buffer.
Example 2
The membrane from Example 1 was placed in an automated imaging
hybridization chamber, to be described momentarily, where it was probed
with a biotinylated oligonucleotide complementary to positions 80 to 110
of the ladder, and then treated with a streptavidin-alkaline phosphatase
conjugate. Complete cycle time was 4.5 hours. The rotation of the inner
cylinder within the hybridization chamber device was set to sweep a bead
of fluid across the convex surface of the membrane, by movement of the
membrane through the fluid, at approximately 20 second intervals.
Hybridization volumes were 50 ml total, and wash volumes were 100 ml each.
Unbound probe was removed by 8 washes with phosphate buffered saline (PBS)
containing 5% SDS. Then a 1/5000 dilution of streptavidin-alkaline
phosphatase (Boehringer Mannheim, 1000 U/ml) was applied in a total volume
of 40 ml of PBS, 5% SDS. The enzyme conjugate was allowed to bind for 45
minutes. Then, unbound conjugate was removed by 1 wash with PBS, 5% SDS; 1
wash with PBS, 1% SDS; 3 washes with PBS; and 3 washes with 0.1M
diethanolamine, pH 10.0, 1 mM MgCl.sub.2, 0.01% sodium azide.
Example 3
The membrane from Example 2 was cut so that each of the three substrates
could be applied to a sequencing ladder. The enzymatic reaction was
started by addition of 1 ml of a fluorogenic alkaline phosphatase
substrate for every 300 cm.sup.2 of membrane. The stock solutions were:
MUFP-50 .mu.g/ml in 0.1M diethanolamine, pH 10; 5MFP-50 .mu.g/ml in 0.1M
diethanolamine, pH 10; and BBTP-600 .mu.g/ml in 2.4 mM diethanolamine, pH
10. The substrate solution was applied as an even coat on the membrane.
In this example, the membranes were imaged outside the chamber, thus
membranes were then placed on glass plates and wrapped with transparent
plastic film. Fluorescence emission was excited by a 458 nm line of an
argon ion laser (Lexel model 96) for 5MFP and BBTP or a long wave UV
mercury lamp for MUFP. Laser light was passed through a lens to widen the
beam distribution and through a 450 nm, 40 nm bandwidth bandpass filter
(Melles Griot). Images were obtained by a cryogenically cooled CH210 CCD
camera (Photometrics Ltd., Tucson, Ariz.) equipped with a 384.times.576
pixel TH7882 (Thomson CSF) CCD array and a 50 mm f/1.2 Pentax camera lens
stopped down to f/22. Images were collected either through a 450 nm, 40 nm
bandwidth bandpass filter (Melles Griot) (MUFP), a 3 mm OG515 (Schott)
color filter (5MFP), or a 560 nm, 10 nm bandwidth bandpass filter (Oriel)
(BBTP). Full length scans (approximately 24 inches) of the sequence
ladders were obtained using TDI mode, which is described in detail below.
During scanning, the blot was moved perpendicularly to the camera on a
translation stage consisting of a Model 506241S rail table, MD series
drive, and a MC3000 controller unit (DAEDAL Inc., Harrison City, Pa.). Two
sets of lanes were acquired at 150 dots per inch in 8 minutes of scan
time. Image data were downloaded from the camera controller frame buffer
to a 286 PC-AT microcomputer, transferred to a Macintosh Quandra for
conversion to a TIFF file and lane-finding, and then to a DEC 5000
workstation for base-calling and sequence analysis, described in detail
below.
In all three cases, sequencing bands were visible to the eye upon
illumination within 15 minutes of substrate application. With both MUFP
and 5MFP, however, the sequencing ladders, though apparent, were visibly
blurred (FIGS. 1A and 1B) even at early time points (65 and 40 minutes,
respectively). The blurring is thought to be due to diffusion or bulk flow
of the fluorescent product, or both. In contrast, BBTP yielded a
relatively sharp ladder, FIG. 1C. Even after prolonged incubation to 20
hours, the sequencing ladder developed with BBTP could be read clearly. It
is thought that the fluorescent product, BBT, interacts with the nylon
membrane, perhaps through a simple hydrophobic interaction, to inhibit
mobility. This property of BBTP, i.e. spatial localization, makes it
clearly the most preferable of the substrates tested for sequence
determination. MUFP and 5MFP appear to provide sufficient fluorescent
product upon dephosphorylation by alkaline phosphatase to be useful for
sequencing applications if a method of spatially localizing the
fluorescent product could be developed. Similarly, other fluorogenic
substrates than the three tested and described here could be used provided
the fluorescent product could be bound to the membrane to yield sharply
defined bands. The large quantities of light emitted using BBTP as
substrate are easily imaged. Further, the fluorescent product, BBT, can be
removed from the membrane by washing with detergent, thus making
subsequent probing feasible. In this instance, BBT was removed from the
membrane by five washes in 5% SDS, 125 mM NaCl, 25 mM sodium phosphate, pH
7.2, for five minutes each at 50.degree. C. Other detergents and wash
conditions may be suitable, also.
One of the advantages of generating blots from DNA sequencing gels from
run-time transfer to the membrane is the constant band-to-band spacing
seen on these blots. The position of sequencing bands on direct transfer
electrophoresis blots is approximately linearly related to fragment
length. Deviations may occur due to compressions, the inverse wedge
profile of the gel, and long term drift in blotting speed. In direct
transfer electrophoresis, the band spacing on the membrane,
.DELTA.S.sub.membrane, is proportional to the blotting speed,
v.sub.blotting, and to the ratio of the band spacing on the gel,
.DELTA.S.sub.gel, to the migration velocity, v.sub.migration. This ratio
is equal to the time interval between the elution of two adjacent bands,
which is approximately constant over a wide range of fragments covering
most of the separated sequencing fragments.
##EQU1##
Direct transfer electrophoresis gives the operator control over the band
spacing on the membrane by choosing the appropriate membrane velocity,
which is an important feature in the case of subsequent CCD based imaging.
It is important to note that while the operator can increase the interband
spacing in direct transfer electrophoresis by increasing the blotting
speed, the electrophoretic resolution is not improved by this process.
Loss of band resolution due to the limited resolution of the imaging
optics is easily prevented by using direct transfer electrophoresis.
Automated Imaging Hybridization Chamber
The automated imaging hybridization chamber is an integrated system for
automatically performing hybridization of nucleic acid probes to
membrane-bound target nucleic acids under controlled conditions. When
certain fluorescent probes are used, such as the enzyme-linked probe
system using BBTP described above, an image of the nucleic acid on the
membrane can be obtained via the transparent window in the outer cylinder
without removing the membrane from the chamber compartment. While the
chamber system described herein is not limited to a single hybridization
chemistry, it was configured specifically for the use of fluorescent probe
systems. It was designed, however, to be flexible enough to permit use
with other systems. Other probes, such as radioactive probes, could be
used in connection with this chamber, but would not utilize the advantage
of imaging with the membrane in the chamber. Further, although the
hybridization chamber system was designed specifically for sequencing
applications, it is capable of handling other membrane-based detection
applications such as colony and plaque hybridizations; Southern, Northern
and Western blot procedures; multiplex genotyping of simple sequence
repeats; sequencing and mapping by hybridization; and dot, slot, and
allele-specific oligonucleotide blot techniques. All of the functions of
the chamber system are computer controlled and fully definable by the
user. The geometry of the chamber device is dictated by a need for the
smallest workable fluid volume, for small air volume, to image the pattern
of nucleic acid on the membrane, and for accurate temperature control
(.+-.2.degree. C.).
Referring now to FIG. 2, the chamber device 10 contains two concentric
nested horizontal cylinders, an inner sealed cylinder 12 having a
continuous cylindrical sidewall 14 and sealed ends 16 and 18, and an outer
cylinder 20 having a cylindrical sidewall 22 containing a visible light
transparent window 24 and entry means 26 for gaining access to the
exterior surface of the sidewall 14 of the inner drum. The end 28 of outer
cylinder 20 is also sealed, whereas end 30 can also constitute the entry
means 26, in which case end 30 is sealable with an "O" ring positioned
between end 30 and sidewall 22. Inner cylinder 12 is removable from outer
cylinder 20 through the entry means 26. The membrane 32 containing bound
nucleic acid to be hybridized is mounted on the exterior surface of the
sidewall 14 of the inner cylinder 12 with stainless steel wire bails (not
shown). The inner cylinder 12 is rotatable on an axle 34 such that there
is a small clearance (e.g., 0.125 inch) between the outside surface of the
sidewall 14 of rotatable inner cylinder 12 and the inside surface of the
sidewall 22 of stationary outer cylinder 20. The space 36 between these
drum surfaces is referred to as the "chamber compartment." The clearance
between the respective sidewalls of the inner and outer cylinders that
defines the chamber compartment 36 should be of sufficient width that the
inner cylinder 12 can rotate within the outer cylinder 20 without the
membrane 32 coming in contact with the interior surface of the outer
cylinder sidewall 22. Disposed on one end of the axle 34 is a driven gear
wheel 38. The mechanism for rotating the inner cylinder 12 includes a
stepper motor 40 which may be disposed on the outside of the outer
cylinder 20 or another stationary object such as a table or machine
chassis (not shown). Coupled to the stepper motor 40 is a driving gear
wheel 42. The driving gear wheel 42 is coupled by a toothed belt 44 to the
driven gear wheel 38 mounted on the axle 34. Thus, when the stepper motor
40 causes the driving gear wheel 42 to rotate, the belt 44 is caused to
rotate the driven gear wheel 38, which in turn rotates the inner cylinder
12 within the outer cylinder 20.
The hybridization process requires various fluids to be brought into
contact with the membrane 32 in the lower portion of chamber compartment
36 and then to be removed by drainage from the chamber compartment 36 and
replaced by other fluids introduced into the chamber compartment 36. It is
crucial that each fluid in the chamber compartment 36 be kept pure and
uncontaminated by other fluids. The various fluids are added to or removed
from the chamber compartment 36 through a number of valves 46 contained in
ports 48 drilled in the bottom of the cylinder 20. Into each port 48 is
threaded a fluid tight spring loaded check valve 46 assembly, FIG. 3.
These valves 46 can be actuated by fluid pressure from pumps that deliver
the solutions to the chamber compartment 36, by means of solenoids, or the
like. The configuration of the valve assemblies 46 with minimal space 47
(FIG. 3) for holding liquid on the chamber side of the valve when the
valve is closed is very significant because of the importance of
minimizing the volume of liquid that would be available to contaminate a
subsequent step of the process. One embodiment of the chamber device 10
contains 20 such ports 48 with associated valve assemblies 46; however,
the number of ports 48 and valve assemblies 46 is variable. The valve
assembly 46 in each port 48 is connected, via a hose connection end 56 of
valve assembly 46, to a hose 58 extending from a fluid delivery module 82,
which will be described in more detail momentari | | |
