U.S. patent number 5,560,811 [Application Number 08/408,683] was granted by the patent office on 1996-10-01 for capillary electrophoresis apparatus and method.
This patent grant is currently assigned to Seurat Analytical Systems Incorporated. Invention is credited to Jonathan Briggs, David W. Hoyt, Randy M. McCormick.
United States Patent |
5,560,811 |
Briggs , et al. |
October 1, 1996 |
Capillary electrophoresis apparatus and method
Abstract
This invention involves method and apparatus for multiplexing
electrophoresis analysis. An array of samples in multi well plates
are simultaneously transferred to an array of electrophoresis
column where electrophoresis is simultaneously carried out followed
by analysis of the columns. The methods and apparatus of this
invention are, for example, useful for DNA analysis, including
sequencing, and for measuring reactions between specifically
binding proteins and their binding partners.
Inventors: |
Briggs; Jonathan (Los Altos
Hills, CA), McCormick; Randy M. (Santa Clara, CA), Hoyt;
David W. (Saratoga, CA) |
Assignee: |
Seurat Analytical Systems
Incorporated (Sunnyvale, CA)
|
Family
ID: |
23617310 |
Appl.
No.: |
08/408,683 |
Filed: |
March 21, 1995 |
Current U.S.
Class: |
204/451;
204/601 |
Current CPC
Class: |
B01L
9/065 (20130101); G01N 27/44743 (20130101); G01N
27/44782 (20130101); G01N 2035/00237 (20130101) |
Current International
Class: |
G01N
27/447 (20060101); G01N 35/00 (20060101); B01D
057/02 (); B01D 059/42 (); C07K 001/26 (); C25B
007/00 () |
Field of
Search: |
;204/299R,180.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Niebling; John
Assistant Examiner: Wong; Edna
Attorney, Agent or Firm: Banner & Allegretti, Ltd.
Claims
What is claimed is:
1. An electrophoresis separation plate comprising:
a) a transportable frame having first and second ends and having an
array of electrophoresis capillaries with first and second ends
mounted respectively between the first and second ends of the
frame;
b) the first end of the frame having a buffer reservoir and an
electrode in the buffer reservoir wherein the first end of the
capillaries are in liquid communication with the buffer reservoir;
and
c) the second end of the frame having a means for placing the
second end of the capillaries in contact with an array of liquid
samples or run buffer which is in contact with electrodes and
wherein there is fluid communication between the buffer reservoir
and the array of samples or the run buffer through the
electrophoresis capillaries and electrical communication between
the electrode in the buffer reservoir and the electrodes in the
samples or run buffer by way of the electrophoresis
capillaries.
2. A system for capillary electrophoresis analysis of an array of
samples in an array of sample containers comprising:
a) means for simultaneously transferring at least a portion of each
sample in the array of samples to a corresponding array of
capillary electrophoresis columns wherein the capillary
electrophoresis columns are in the electrophoresis plate of claim
1;
b) means for simultaneously conducting capillary electrophoresis
separations on the array of transferred samples in the capillary
electrophoresis columns; and
c) means for analyzing capillary electrophoresis separations from
(b).
3. An apparatus for processing an array of samples from an array of
sample wells comprising:
a) a sample handling plate which defines an array of sample
handling plate wells wherein each well has a sipper capillary in
liquid communication with the sample handling plate well to provide
an array of sipper capillaries and wherein a porous matrix is
interposed between each sipper capillary and each sample handling
plate well, wherein the array of sipper capillaries will
simultaneously wick an array of samples from an array of sample
wells;
b) a base plate which defines an opening to receive the sample
handling plate and defines an inner chamber to means associated
with the base plate and sample handling plate to seal the inner
chamber and provide a sealed inner chamber; and
c) means for pressurizing and evacuating the sealed inner chamber
to move liquid on either side of the porous matrix to the other
side of the porous matrix.
4. Electrophoresis separation plate comprising:
a frame having a buffer reservoir at one end and a plurality of
sample sites at the other end and having an electrode at each
end,
a means for mounting capillary electrophoresis columns on the frame
so that there is electrical communication between the electrodes
and fluid communication between the sample sites and reservoir when
there is fluid in the sample sites, capillary electrophoresis
columns and buffer reservoir.
5. A method for analysis of an array of samples in an array of
sample containers by capillary electrophoresis comprising:
a) providing an array of samples in an array of sample wells;
b) simultaneously transferring at least a portion of each sample in
the array of sample wells to a corresponding array of capillary
electrophoresis columns wherein the capillary electrophoresis
columns are in the electrophoresis separation plate of claim 1;
c) simultaneously conducting separation of the transferred samples
by capillary electrophoresis; and
d) analyzing the capillary electrophoresis separations of (c).
6. A method for analysis of an array of samples in an array of
sample containers by capillary electrophoresis comprising:
a) providing the array of samples in an array of sample wells;
b) simultaneously acquiring an aliquot of each sample from the
array of samples with a sample handling plate having an array of
sample handling plate wells with sipper capillaries;
c) transferring the sample handling plate to a base plate which
provides for simultaneously processing and then presenting the
samples for capillary electrophoresis;
d) simultaneously transferring at least a portion of the presented
samples to an array of capillary electrophoresis columns;
e) simultaneously conducting separations by capillary
electrophoresis of the presented transferred samples; and
f) analyzing the capillary electrophoresis separation of e.
7. A system according to claim 2 wherein the array of samples
conforms to the format of a 96, 192 or 384 well plate.
8. A separation plate according to claim 1 wherein the plate
contains 8, 12, 16 and 24 electrophoresis capillaries spaced apart
to match the spacing in a row or columns of 8, 12, 16 or 24 wells
in a 96, 192, or 384 well plate.
9. The apparatus of claim 3 wherein the sipper capillaries of the
sample handling plate wells are in an array which conforms to a
format of a 96, 192 or 384 well plate.
10. The apparatus of claim 3 wherein the means for pressurizing and
evacuating is programmed to alternately pressurize and evacuate the
inner chamber to mix reagents added above the porous matrix with
sample below the porous matrix.
11. The method of claim 5 wherein the array of samples is nucleic
acid of varying length.
12. The method of claim 5 wherein the array of samples is a
specifically binding protein, its binding partner or the complex of
the two.
13. The method of claim 6 wherein one or more reagents are added to
the sample handling plate wells and mixed with the samples.
14. The method of claim 6 wherein the array of electrophoresis
columns are rinsed for reuse to separate additional samples.
15. The electrophoresis separation plate of claim 1 wherein there
is provided adjacent to sample handling plate wells a buffer wash
well and a run buffer well.
16. The electrophoresis separation plate of claim 1 wherein
electrodes are mounted in the frame near the second end of the
capillaries so that when the capillaries are in the samples or the
run buffer there is electrical communication between the electrode
in the buffer reservoir and the electrodes near the
capillaries.
17. The apparatus of claim 3 wherein the porous matrix is a
membrane.
Description
BACKGROUND OF THE INVENTION
A. Field Of the Invention
This invention is in the field of separation of biomolecules and,
in particular, separations by capillary electrophoresis and the use
of the capillary electrophoresis to detect such molecules.
B. Background of the Prior Art
Electrophoresis is a separation process in which molecules with a
net charge migrate through a medium under the influence of an
electric field. Traditionally, slab gel electrophoresis has been a
widely used tool in the analysis of genetic materials. See, for
example, G. L. Trainor, Anal. Chem., 62, 418-426 (1990). Capillary
electrophoresis has emerged as a powerful separation technique,
with applicability to a wide range of molecules from simple atomic
ions to large DNA fragments. In particular, capillary
electrophoresis has become an attractive alternative to slab gel
electrophoresis for biomolecule analysis, including DNA sequencing.
See, for example, Y. Baba et at., Trends in Anal. Chem., 11,
280-287 (1992). This is generally because the small size of the
capillary greatly reduces Joule heating associated with the applied
electrical potential. Furthermore, capillary electrophoresis
requires less sample and produces faster and better separations
than slab gels.
Currently, sophisticated experiments in chemistry and biology,
particularly molecular biology, involve evaluating large numbers of
samples. For example, DNA sequencing of genes is time consuming and
labor intensive. In the mapping of the human genome, a researcher
must be able to process a large number of samples on a daily basis.
If capillary electrophoresis can be conducted and monitored
simultaneously on many capillaries, i.e., multiplexed, the cost and
labor for such projects can be significantly reduced. Attempts have
been made to sequence DNA in slab gels with multiple lanes to
achieve multiplexing. However, slab gels are not readily amenable
to a high degree of multiplexing and automation. Difficulties exist
in preparing uniform gels over a large area, maintaining gel to gel
reproducibility and loading sample wells. Furthermore, difficulties
arise as a result of the large physical size of the separation
medium, the requirements for uniform cooling, large amounts of
media, buffer, and samples, and long run times for extended reading
of nucleotide sequences. Unless capillary electrophoresis can be
highly multiplexed and multiple capillaries run in parallel, the
advantages of capillary electrophoresis cannot produce substantial
improvement in shortening the time needed for sequencing the human
genome.
Capillary electrophoresis possesses several characteristics which
makes it amenable to this application. The substantial reduction of
Joule heating per lane makes the overall cooling and electrical
requirements more manageable. The cost of materials per lane is
reduced because of the smaller sample sizes. The reduced band
dimensions are ideal for excitation by laser beams, as well as
focused extended sources, and for imaging onto array detectors or
discrete spot detectors. The concentration of analyte into such
small bands results in high sensitivity. The use of
electromigration injection, i.e., applying the sample to the
capillary by an electrical field, provides reproducible sample
introduction with little band spreading, minimal sample
consumption, and little labor.
Among the techniques used for detecting target species in capillary
electrophoresis, laser-excited fluorescence detection so far has
provided the lowest detection limits. Therefore, fluorescence
detection has been used for the detection of a variety of analytes,
especially macromolecules, in capillary electrophoresis. For
example, Zare et al. (U.S. Pat. No. 4,675,300) discusses a
fluoroassay method for the detection of macromolecules such as
genetic materials and proteins in capillary electrophoresis. Yeung
et al. (U.S. Pat. No. 5,006,210) presented a system for capillary
zone electrophoresis with laser-induced indirect fluorescence
detection of macromolecules, including proteins, amino acids, and
genetic materials.
Systems such as these generally involve only one capillary. There
have been attempts to implement the analysis of more than one
capillary simultaneously in the electrophoresis system, but the
number of capillaries has been quite small. For example, S.
Takahashi et al., Proceedings of Capillary Electrophoresis
Symposium, December, 1992, referred to a multi-capillary
electrophoresis system in which DNA fragment samples were analyzed
by laser irradiation causing fluorescence. This method, however,
relies on a relatively poor focus (large focal spot) to allow
coupling to only a few capillaries. Thus, this method could not be
applied to a large number of capillaries. This method also results
in relatively low intensity and thus poor sensitivity because of
the large beam. Furthermore, detection in one capillary can be
influenced by light absorption in the adjacent capillaries, thus
affecting accuracy due to cross-talk between adjacent
capillaries.
Attempts have been made to perform parallel DNA sequencing runs in
a set of up to 24 capillaries by providing laser-excited
fluorometric detection and coupling a confocal illumination
geometry to a single laser beam and a single photomultiplier tube.
See, for example, X. C. Huang et at., Anal. Chem., 64, 967-972
(1992), and Anal. Chem., 64, 2149-2154 (1992). Also see U.S. Pat.
No. 5,274,240. However, observation is done one capillary at a time
and the capillary bundle is translated across the
excitation/detection region at 20 mm/see by a mechanical stage; the
capillaries in this system are not transportable to a different
site for measurement.
There are features inherent in the con focal excitation scheme that
limit its use for very large numbers of capillaries. Because data
acquisition is sequential and not truly parallel, the ultimate
sequencing speed is generally determined by the observation time
needed per DNA band for an adequate signal-to-noise ratio.
Moveover, the use of a translational stage can become problematic
for a large capillary array. Because of the need for translational
movement, the amount of cycling and therefore bending of the
capillaries naturally increases with the number in the array. It
has been shown that bending of the capillaries can result in loss
in the separation efficiency. This is attributed to distortions in
the gel and multipath effects. Sensitive laser-excited fluorescence
detection also requires careful alignment both in excitation and in
light collection to provide for efficient coupling with the small
inside diameter of the capillary and discrimination of stray light.
The translational movement of the capillaries thus has to maintain
stability to the order of the confocal parameter (around 25 .mu.m)
or else the cylindrical capillary walls will distort the spatially
selected image due to misalignment of the capillaries in relation
to the light source and photodetector. In addition, long
capillaries provide slow separation, foul easily, and are difficult
to replace.
U.S. Pat. No. 5,324,401 to Yeung et al. describes a multiplexer
capillary electrophoresis system where excitation light is
introduced through an optical fiber inserted into the capillary. In
this system the capillaries remain in place, i.e. in the buffer
solutions when the capillaries are read.
U.S. Pat. No. 5,332,480 (Dalton et al.) describes a multiple
capillary electrophoresis device for continuous batch
electrophoresis.
U.S. Pat. No. 5,277,780 (Kambara) describes a two dimensional
capillary electrophoresis apparatus for use with a two dimensional
array of capillaries for measuring samples, such as DNA samples, in
an array of test wells.
U.S. Pat. No. 5,338,427 (Shartle et at.) describes a single use
capillary cartridge having electrically conductive films as
electrodes; the system does not provide for multiplexed sampling,
sample handling, and electrophoresis.
U.S. Pat. Nos. 5,091,652 (Mathies et at.) and 4,675,300 (Zare et
al.) describe means for detecting samples in a capillary.
U.S. Pat. No. 5,372,695 (Demorest) describes a system for
delivering reagents to serve a fix capillary scanner system.
Numerous examples of sample handling for capillary electrophoresis
are known. For example, James in U.S. Pat. No. 5,286,652 and
Christianson in U.S. Pat. No. 5,171,531 are based on presenting a
single vial of sample to a single separation capillary for a
sequential series of analyses.
Goodale in U.S. Pat. No. 5,356,625 describes a device for
presentation of a tray of 7 vials of sample to an array of seven
capillaries for the sample injection process.
Carson in U.S. Pat. No. 5,120,414 describes injection of a sample
contained within a porous membrane onto a single-capillary
electrophoresis device. The end of the capillary must be in
intimate contact with the porous membrane to affect sample
introduction into the capillary.
In contrast, the present invention provides short disposable
capillaries mounted in a frame which is integral with a liquid
handling system. This system permits rapid multiplexed approach to
capillary electrophoresis.
Numerous examples of multi-well devices with integral membranes are
known (e.g. Mann in U.S. Pat. No. 5,043,215, Matthis in U.S. Pat.
No. 4,927,604, Bowers in U.S. Pat. No. 5,108,704, Clark in U.S.
Pat. No. 5,219,528). Many of these devices attach to a base unit
which can be evacuated, drawing samples through the membrane for
filtration.
Numerous examples of multi-channel metering devices such as
multi-channel pipettes are known. One example is described in a
device by Schramm in U.S. Pat. No. 4,925,629, which utilizes an
eight-channel pipette to meter samples/reagents to/from multi-well
plates. A second example is a 96-channel pipetting device described
by Lyman in U.S. Pat. No. 4,626,509. These devices use positive
displacement plungers in corresponding cylinders to draw in and
expel liquid in the sampling/metering step.
Finally, Flesher in U.S. Pat. No. 5,213,766 describes a 96-channel
device which contains flexible "fingers" which can be deformed out
of a common plane; each "finger" can be deflected into a well of a
multi-well plate to acquire a small aliquot of sample by one of
several mechanisms.
The present invention differs in that it provides for
simultaneously sampling of an array as samples; simultaneously
handling the samples and presenting an array of the samples for
capillary electrophoresis; simultaneously transferring the array of
presented samples to an array of capillaries; and simultaneously
conducting separations in the capillary electrophoresis
columns.
SUMMARY OF THE INVENTION
The present invention encompasses methods and apparatus for
simultaneously transferring samples from an array of sample holders
to an array of capillary eletrophoresis columns, simultaneously
conducting electrophoresis, and analyzing the capillary
electrophoresis columns.
The invention encompasses a system for multiplexing capillary
electrophoresis analysis of multiple samples comprising:
a) a means for simultaneously acquiring an array of aliquots of
sample from an array of samples in sample containers;
b) a means, in combination with means (a), for simultaneously
processing the array of samples to provide an array of processed
samples and presenting the array of processed samples for capillary
electrophoresis;
c) means for simultaneously transferring an array of processed
samples to an array of capillary electrophoresis columns;
d) means for simultaneously conducting capillary electrophoresis on
the array of the capillary electrophoresis columns from (c);
and
e) means for analyzing capillary electrophoresis columns from
(d).
The invention also encompasses an electrophoresis separation plate
comprising:
a) a frame having a first and second end and having an array of
electrophoresis capillaries with first and second ends mounted
respectively between the first and second end of the frame;
b) the first end of the frame having a buffer reservoir and an
electrode in the buffer reservoir wherein the first end of the
capillaries are in liquid communication with the buffer
reservoir;
c) the second end of the frame having a means for placing the
second end of the capillaries in contact with an array of liquid
samples or run buffers which is in contact with an array of
electrodes and wherein there is fluid communication between the
buffer reservoir and the sample or run buffer through the
capillaries and electrical communication between the electrode in
the buffer reservoir and the electrodes in the samples or run
buffer by way of the electrophoresis capillaries.
The invention further encompasses an apparatus for processing
samples from an array of sample wells comprising:
a) a sample handling plate which defines an array of sample
handling plate wells wherein each well has a sipper capillary in
liquid communication with the sample handling plate well to provide
an array of sipper capillaries and wherein a porous matrix is
interposed between each sipper capillary and the sample handling
plate well, wherein the array of sipper capillaries will
simultaneously wick samples from an array of sample wells;
b) a base plate which defines an opening to receive the sample
handling plate and defines an inner chamber and means associated
with the base plate and sample handling plate to seal the inner
chamber and provide a sealed inner chamber;
c) means for pressurizing and evacuating the sealed inner chamber
to move liquid on either side of the porous matrix to the other
side of the porous matrix.
The invention also encompasses, in another embodiment, a
electrophoresis separation plate comprising a frame having a
reservoir at one end and a plurality of sample sites at the other
end and having an electrode at each end and a means for mounting
capillary electrophoresis columns on the frame so that there is
electrical communication between the electrodes and fluid
communication between the sample site and reservoir when there is
fluid in the sample site and reservoir. The electrophoresis plate
may be transportable so that it can be moved to various locations
and stored.
The invention further involves a method for multiplexed analysis of
multiple samples by capillary electrophoresis comprising:
a) providing the samples in an array of sample wells;
b) simultaneously sampling the samples with a sample handling plate
having an array of sample handling plate wells with sipper
capillaries;
c) transferring the sample handling plate to a base plate which
provides for simultaneously processing and presenting the samples
for electrophoresis;
d) simultaneously transferring pressurized sample to an array of
capillary electrophoresis columns;
e) simultaneously conducting separation by electrophoresis;
f) analyzing the capillary electrophoresis columns.
The electrophoresis plate comprises a frame with at least one
reservoir at one end. The electrophoresis plate has a means for
mounting a plurality of capillary electrophoresis columns, having
first and second ends, on the frame so that there is fluid
communication between the reservoir and the first end of the
plurality of capillaries, when there is fluid in the reservoir, and
a plurality of sample introduction sites near or at the second end
of the capillaries. There is a means to make electrical connection
to each end of the frame, with at least one common electrode in the
reservoir in the frame at the first end of the capillaries and at
least one electrode in a second common reservoir in the base plate
in fluid communication with the plurality of sample sites at the
second end of the capillaries. Them is a plurality of electrodes
electrically connected in parallel and positioned in each of the
sample introduction sites at the second end of the capillaries.
This arrangement of electrodes provides for electrical
communication between the electrodes at each end of the frame and
the capillaries, when there is fluid in the reservoirs, capillaries
and the sample introduction sites.
The invention includes methods for analyzing samples of substances
which are separable by electrophoresis providing a transportable
electrophoresis plate having a plurality of electrophoresis
columns, simultaneously transferring sample from the multiple
samples to the electrophoresis columns in the transportable plate,
simultaneously conducting electrophoresis on the columns in the
plate to separate the substance to be analyzed, moving the
transportable plate to a location for analysis, and analyzing the
substance(s) to be analyzed which are separated in the
electrophoresis columns in the transportable plate. The capilllary
columns may be dynamically analyzed by detecting a separated band
as it moves past a stationary analysis system, or the columns may
be scanned by a moveable analysis system, on imaged by an array
detector.
The present invention has many advantages over conventional
electrophoresis technology. For example, the confirmation of proper
amplification of nucleic acids by conventional separation on gels
takes hours; whereas, the system of this invention can do
confirmation in about 10 minutes. Conventional gel separations are
manually intensive. The invention only requires sample
introduction, with the separation, detection and analysis being
automatic. The invention system will perform confirmations in
parallel. For example, 96 samples can be processed on a standard
gel, but only with compromises in resolution and sensitivity.
The invention system can resolve a difference of 2-3 base pairs
among double stranded DNA fragments of 200-300 bps. If 96 samples
are run in parallel on a single standard gel, resolution for the
same range of targets would be 5 to 10 fold poorer. Working with
samples of unpurified PCR-amplified DNA, the present system found
multiple amplified targets in several samples where the standard
gel methods only detected a single band.
The present invention system typically requires only 1 to 5% of the
amount of sample used on a standard gel because of the high
sensitivity and hence less amplified material is required, reducing
reagent cost and/or amplification time.
The capability to add a common reagent to multiple samples, mix and
react means that the primary amplification step could be done on
the sample handling plate, with an approximately designed
thermocycler, with the benefits of amplification done in discrete
small volumes, in parallel with precise timing, with a minimum of
carry-over and cross contamination.
This invention has advantages with regard to quantitation of
amplified nucleic acids. After amplification, the standard methods
are patterned after conventional immunoassays requiring solid phase
reactions (on the surface of 96-well plates or on beads), followed
by washes for separation and subsequent reactions for signal
generation. The whole procedure takes several hours, with many
steps, and with marginal sensitivity and precision.
The present invention system can perform a quantitative
determination in a much simpler format. The target is amplified
with sequence specificity in the standard way; that is, for PCR,
specificity is derived from the primers and for LCR specificity is
derived from the ligation of adjacent hybridized probes. Then the
amplified material is separated on columns and the amount of target
is measured at the anticipated position on the column, based on the
size of the target. That is, one quantitates the amount of target,
in addition to obtaining a size-based confirmation without the need
for any solid phase reactions. If additional sequence specificity
is required, then an additional hybridization step could be
incorporated into the procedure.
Since reactions are in the liquid phase, the invention system
provides greater speed, greater specificity and less background
biochemical noise and quantitation is achieved in 10's of minutes
instead of hours.
The present invention system has demonstrated the detection of as
little as 8 million DNA molecules (300 to 1300 bps) in the sample
and therefore has high sensitivity. Because of the high sensitivity
of the present system, less biological amplification is required.
For example, 20 PCR cycles operating at optimum efficiency will
produce 1 million molecules, starting from a single target molecule
in the sample. Conventional quasi-quantitative methods typically
require 30 to 40 cycles to produce enough target for reliable
detection. However, the biological gain per cycle decreases as one
amplifies at the higher cycle numbers (experts agree that such
variability can occur at greater than about 20 cycles). This
variability is a primary cause for the lack of assay precision in
the conventional methods. The present invention system only
requires 2 .mu.l of sample, reducing the amount of primers, bases,
enzymes, etc. required for the amplification step.
The capability to add a common reagent to multiple samples, mix and
react means that the primary amplification step could be done on
the sample handling plate, with an appropriately designed
thermo-cycler, with the benefits of amplification done in discrete
small volumes, in parallel with precise timing, with a minimum of
carry-over and cross contamination.
Many of these advantages are also achieved with regard to
conventional binding assays such as ELISA's. For example,
quantitation can be done in 10's of minutes on 2 .mu.l sample
volumes. The capability to add a common reagent to multiple samples
in, for example, a 96 well or a multi well plate, mix, and react on
the sample handling plate means that a primary reaction step (e.g.
displacement of a common ligand to a receptor) can be done in
discrete small volumes, in parallel with precise timing, with a
minimum of carry-over and cross contamination, and without
contamination of the starting material (e.g. any array of compound
libraries).
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a perspective view of the electrophoresis separation
plate.
FIG. 2 is a cross-sectional view of the separation plate through a
capillary.
FIG. 3a shows an exploded sectional view of the well with the well
electrode and capillary positioned in the well.
FIG. 3b shows a sample injected into the well.
FIG. 3c shows the well with buffer and sample in the well.
FIG. 3d shows the well when the buffer has diluted the sample.
FIG. 4a-c shows a schematic of how the components of the system
interact.
FIG. 5a shows a cross-sectional view of several sample handling
plate wells.
FIG. 5b shows a schematic of liquid flow from sipper capillary to
sample handling plate well.
FIG. 6 shows flow of sample into sipper capillary.
FIG. 7a-7e shows the flow of sample and reagent mixing.
FIG. 8 shows sample handling plate with sample handling plate well
and waste and primary buffer wells.
FIG. 9 shows top plan view of a preferred separation plate.
FIG. 10 shows a cross-sectional view of the preferred separation
plate through a capillary.
FIG. 11 shows a schematic diagram of the optical system for reading
capillaries.
FIG. 12 graph of Msp pBR322 separation.
FIG. 13 shows a trace of a separation of single stranded DNA
fragments differing by one base.
FIG. 14 shows separation of unknown DNA samples derived from
PCR.TM. amplification.
FIGS. 15A and B shows separation of a protein/binding partner
complex.
FIG. 16 shows separation of a displaced labeled peptide versus
concentration of a competitor.
FIG. 17 illustrates the limits of detection of DNA.
FIG. 18 shows the efficiency of mixing of two samples in the sample
handling plate.
FIGS. 19A-F illustrates the reproduceability of simultaneous
sipping, sample handling, electrophoresis and then analysis of DNA
samples.
DETAILED DESCRIPTION OF THE INVENTION
The invention includes as shown in FIGS. 1 and 2 an elongated
electrophoresis separation plate 1 which has a plurality of sample
wells 2 at one end and a common buffer reservoir 3 at the other
end. A first master electrode 5 is electrically connected to a cell
electrode 6 in the sample wells 2. A second master electrode 7 is
in the common buffer reservoir 3. Capillary electrophoresis columns
8 are mounted in the plate 1 so that there is electrical
communication between the first master electrode 5 by way of the
capillary electrophoresis column 8 when the sample wells 2 and the
reservoir 3 are filled with electrically conductive liquid. In
operation, current between the master electrodes permits
electrophoresis of the sample from the sample well 2 to the
reservoir 3.
FIG. 3a is a partial sectional view through a sample well 2 showing
the well electrode 6 and capillary electrophoresis column 8. FIG.
3b illustrates the injection of a sample 15 so that there is liquid
communication between the capillary 8 and cell electrode 6. The
sample is loaded on the capillary via electromigration injection
and then the residual sample in well 2 is diluted with buffer 19
before the electrophoresis process takes place as shown in FIG. 3d.
In an alternative implementation, FIG. 3c shows a mechanical liquid
transfer system where about 4 .mu.l of buffer 17 is first added to
establish liquid communication between the capillary 8 and cell
electrode 6. Then 4 .mu.l of sample 18 contained within a pipetter
tip is placed in contact with the buffer 17 and sample is loaded on
the column via electromigration injection. After removing the
pipette tip, the well is then filled with buffer 19 as shown in
FIG. 3d and the electrophoresis is conducted.
Sample may be made available for injection from the introduction
site to the capillary such that only small aliquots of the primary
sample are required at the introduction sites, quantitative
injection is possible, and there is a minimum of carryover from one
injection to another (if the plates are reused). Such sample
introduction may be accomplished by a variety of means,
including:
a) physically moving the capillaries relative to the introduction
sites and immersing the tips of the capillaries into the respective
sample aliquots positioned within the introduction sites. In this
embodiment the plurality of sample introduction sites might be
constructed on a separate disposable part, which moves into
position for sample introduction, and then is disposed.
b) physically moving the capillaries relative to the introduction
sites and bringing the tips of the capillaries to the close
proximity of the sample aliquots in the introduction sites.
c) each introduction site is permanently immediately adjacent to
the respective capillary tip.
Sample injection may be accomplished, simultaneously and in
parallel, for the plurality of capillaries by a variety of means,
including:
a) electroinjection, under the action of an electric field due to a
voltage difference applied to the appropriate electrodes.
b) pressure injection, under the action of a pressure or suction
applied to the fluid at one or both ends of the capillaries.
With regard to electrodes, some or all of the electrodes may be
within the sample handling plate or within the electrophoresis
separation plate, with external connections to power supplies, or
some or all of the electrodes might be on a separate part (e.g.
built into the injection and separation station), such that the
electrodes can be immersed into the appropriate fluid reservoirs at
the time of injection or separation. The electrodes may also be
integral with the separation plate. They may be strip metal
electrodes formed in a stamping process or chemical etching
process. The electrodes may be wires or strips either soldered or
glued with epoxy and can be made of conductive materials such as
platinum, gold, copper, carbon fibers and the like. Electrodes
could be integral with the sample handling plate formed by silk
screening process, printing, vapor deposition, electrode-less
plating process, etc. Carbon paste, conductive ink, and the like
could be used to form the electrode.
Those skilled in the electrophoresis arts will recognize a large
number of capillaries useful for practicing this invention. For
example, fused silica is used with an outside coating of polyimide
for strengthening, with inside bore ID's from 10 to 200 microns,
more typically from 25 to 100 microns, and OD's greater than 200
microns. Internal coating may be used to reduce or reverse the
electroosmatic flow (EOF). The simplest "coating" involves running
at a low pH such that some of the silanol negative charge is
neutralized. Other coatings include: silylation, polyacrylamide
(vinyl-bound), methylcellulose, polyether, polyvinylpyrrolidone
(PVP), and polyethylene glycol. Other materials used for
capillaries include quartz, Pyrex.TM. and Teflon.TM..
Conventional buffers include the Good's buffers (HEPES, MOPS, MES,
Tricine, etc.), and other organic buffers (Tris, acetate, citrate,
and formate), including standard inorganic compounds (phosphate,
borate, etc.). Two preferred buffered systems are:
i) 100 mM sodium phosphate, pH 7.2
ii) 89.5 mM tris-base, 89.5 mM Boric acid, 2 mM ETDA, pH 8.3.
Buffer additives include: methanol, metal ions, urea, surfactants,
and zwitterions interculating dyes and other labeling reagents.
Polymers can be added to create a sieving buffer for the
differential separation of DNA based on fragment length. Examples
of polymers are: polyacrylamide (cross-linked or linear), agarose,
methylcellulose and derivatives, dextrans, and polyethylene glycol.
Inert polymers can be added to the separation buffer to stabilize
the separation matrix against factors such as convection
mixing.
Those skilled in the electrophoresis arts will recognize a wide
range of useable electric field strengths, for example, fields of
10 to 1000 V/cm are used with 200-600 V/cm being more typical. The
upper voltage limit for the commercial systems is 30 kV, with a
capillary length of 40-60 cm, giving a maximum field of about 600
V/cm. There are reports of very high held strengths (2500-5000
V/cm) with short, small bore (10 microns) capillaries
micro-machined into an insulating substrate.
Normal polarity is to have the injection end of the capillary at a
positive potential. The electroosmotic flow is normally toward the
cathode. Hence, with normal polarity all positive ions and many
negative ions will run away from the injection end. Generally, the
"end-of-capillary" detector will be near the cathode. The polarity
may be reversed for strongly negative ions so that they run against
the electroosmotic flow. For DNA, typically the capillary is coated
to reduce EOF, and the injection end of the capillary is maintained
at a negative potential.
FIGS. 4a-c show the interaction of various parts of the
electrophoresis system. FIG. 4a shows the sample handling plate 71
with an array of sample handling wells 74 with an corresponding
array of sipper capillaries 82. The array of sipper capillaries is
aligned with wells of a multiwell plate which contain samples 85.
When the sipper capillaries 82 are in the sample, an aliquot of
sample is transferred to the sipper capillary by wicking action.
The sample handling plate 71 is then moved to base plate 72, as
shown in FIG. 4b. Sample handling plate 71 and base plate 72 fit
together to form a sealed inner chamber 69 which can be pressurized
or evacuated through port 73. In this way, the samples in
capillaries 82 can be manipulated and eventually presented in
sample handling wells 74 for electrophoresis. FIG. 4c shows how the
electrophoresis separation plate(s) 100 containing an array of
electrophoresis capillaries 101 are aligned with the sample
handling plate wells 74.
FIG. 5a shows a cross-sectional view through several wells 74. The
sample handling plate 71 is assembled from a sampling block 75
which defines the funnel shaped base wells with openings 77. Mixer
block 78 has passages 79 which are aligned with openings 77. The
mixer block 78 and sampling block 75 are separated by a porous
matrix such as membrane 80. Aligned with mixer block 78 is sipper
block 81 with sipper capillaries 82. 82a is filled with sample and
82b is not. The sipper block 81, mixer-block 78, and sampling block
75 are fastened together so that a channel a-b is defined which is
interrupted by the membrane 80 as shown in FIG. 5b. Membrane 80 is
typically made of a wide variety of porous matrix materials where,
for most applications the porous matrix materials should have
little or no affinity for sample. Useful porous matrix materials
include membrane materials such as regenerated cellulose, cellulose
acetate, polysulfone, polyvinylidine fluoride, polycarbonate and
the like. For DNA samples, a cellulose acetate membrane such as
that available from Amicon is useful. For protein samples, a
membrane composed of polysulfone such as those available from
Amicon or Gelman is useful.
FIG. 6 illustrates the flow of sample 85 from a well of a multiwell
plate 86 into sipper capillary 82. Thus the ends of the array of
sipping capillaries 82 on sample handling plate 71 are dipped into
samples contained in an array of samples such as a 96 well plate
and the samples are metered into the sipping capillaries by
capillary action. At this point, the sample handling plate 71 with
its sipper capillaries filled with samples is placed on base 72 to
form a sealed inner chamber 69, FIG. 4b.
FIG. 7a-7e illustrates the flow of sample in response to
pressurization and evacuation of the inner sealed chamber 69
through port 73. For example, a positive pressure moves the sample
from the sipping capillary 82 to the area below the membrane, as
shown in 7a, through the membrane, and into the well 74 above the
membrane in plate 71. Reagents 67 can be added to the wells 74 as
shown in FIG. 7b and the reagent and sample can be mixed as shown
in 7c and 7d by forcing the sample and reagent back and forth
through the membrane 80 in response to pressurization and
evacuation of the inner sealed chamber 69. Finally, the mixed
sample 68 is presented in well 74 for injection into an
electrophoresis column as shown in 7e.
FIG. 8 illustrates the sample well 74 flanked with a well for waste
electrophoresis buffer 90 from a previous separation and a well for
fresh run buffer 91 which is deposited from a capillary 105 during
a flushing before injecting presented sample from the sample
handling well into the capillary 105 and then used during the
electrophoresis separation. The capillary 105 addresses the waste
90, buffer 91 and sample 74 positions by moving the separation
plate with respect to the sample handling plate for
electrophoresis.
FIG. 9 shows the electrophoresis separation plate 100 having 8
capillaries 101 mounted on a frame 102; upper buffer reservoir 103
provides buffer to the capillaries 101. Orientation notch 113
provides a means for aligning the separation plate for transferring
sample or reading columns. Electrode 104 and an electrode at the
injection end of each of the separation capillary 105 provide for
electrical communication through the buffer. FIG. 10 is a
cross-sectional view through a capillary.
Those skilled in the arts will recognize that parts such as the
sample handling plate, base plate and frame of the separation plate
can be machined or molded from chemical resistant plastics such as
polystyrene or the like.
Thus, in operation, samples from an array of samples 85 such as a
multiwell plate are wicked into an array of sipping capillaries 82
of the sample handling plate 71. The sample handling plate 71 is
placed on base 72 and the sample is manipulated by pressurizing the
chamber 69 defined by the sample handling and base plates and
finally moved to the base plate wells 74 for presentation to the
capillaries in the separation plate 100. However, prior to
transferring the sample to the capillary, the capillaries are
washed with buffer and primed with buffer. Samples are injected
into the capillaries and electrophoresis is conducted in the
capillaries in the separation plate. When the electrophoresis is
finished, the separation plate may be moved to an analysis station.
The over all scheme is shown in FIGS. 4a-c. After electrophoresis,
the separation plate can be stored or read as shown in FIG. 11.
Capillary electrophoresis columns can be analyzed in variety of
ways including the methods shown in U.S. Pat. Nos. 4,675,300,
4,274,240 and 5,324,401. The sample injection and separation are
conducted in one location and the plate may be transported to a
different location for analysis. FIG. 11 shows a block diagram of
one optical system for reading the capillaries. Power supply 30
energizes the photomultiplier tube 31. Power supply 32 energizes a
75 watt Xenon lamp 75. Light from the lamp 75 is condensed by
focusing lens 34 which passes light to the excitation filter 35. A
dichroic mirror 36 directs excitation light to microscope objective
37. The separation plate 100 with capillaries 101 is mounted on a
rectilinear scanner to pass the capillaries over the light from the
microscope objective 37.
Those skilled in this art will recognize that the above liquid
handling system provides for simultaneous and quantitative sampling
of a large array of samples by sipping from the 96, 192 or 384-well
plates or arrays of microtubes with an array of sipper capillaries.
It provides for mixing separate aliquots in the .mu.l range by
cycling the aliquots back and forth through a porous matrix such as
a membrane. The invention provides an array of addition and mixing
sites for the simultaneous addition and mixing of reagents for
achieving either a constant or a gradient of mixed material across
the array and for precisely controlling for the simultaneous
starting or stopping of reactions.
Use of activated membranes in the base plate provides for selective
removal of some components of the sample of reaction mixture prior
to injection. For example, an ultrafiltration membrane may be used
for the removal of high molecular weight constituents or an
affinity membrane, (e.g., protein-A membranes) for the removal of
IgG or lectin-membranes for removal of carbohydrates or membranes
with a specific antibody directed against biopharmaceutical product
to remove the great excess of product for impurity analysis for
process and quality control.
EXAMPLE I
This example illustrates separation and detection of MspI pBR322
fragments under the following conditions:
______________________________________ SEPARATOR BREADBOARD:
SEPARATION PLATE AS SHOWN IN FIG. 1. CAPILLARIES TYPE: 30 MICRON ID
FUSED SILICA DERIVATIZED WITH 3.5% LINEAR POLY- ACRYLAMIDE. LENGTH:
109 MM WINDOW LOCATION: 10-100 MM; BARE SILICA CLEANING PROCEDURE
FLUSHED WITH WATER, THEN BUFFER SAMPLES: MSP I PBR 322 DNA 5 uG/ML
IN 0.5 X TBE LOADED 5 IN CYLINDRI- CAL TEFZEL WELL FOR INJECTION
DETECTOR NIKON EPI- FLUORESCENCE MICROSCOPE PTI ANALOGUE PM SYSTEM
GAIN: 0.01 .mu.A/VOLT TIME CONSTANT: 50 MSEC PMT VOLTAGE: 1000 V
LAMP XENON IRIS: Open N.D. FILTERS: None FILTER SET: G2A CUBE
(ETBR): (Dich 580 nm, Exc 510-560 nm, Em 590 nm) FOCUS & SLITS:
focus on inner bore, set slits +25 .mu.m on either side of bore
diameter DETECTOR POSITION: 45 mm scan of capillary along x axis
using detection system illustrated in FIG. 11. OBJECTIVE: 10X DATA
SYSTEM: PE NELSON MODEL 1020 DATA COLL. RATE: 20-40 HZ BUFFERS
SAMPLE: 0.5 X TE CAPILLARY (pre load): STOCK NUCLEOPHOR .TM. BUFFER
+ 2.5 .mu.g/mL ethidium bromide END CHAMBERS: At ground: ethidium
bromide sieving buffer, not plugged (vented to atm pres.); at
Sample end: 2.5 ug/ml ethidium bromide in TBE electrolyte INJECTION
METHOD: Electrokinetic, capillary cassette in horizontal position
TIME: 10 sec VOLTAGE: 3.33 kV SAMPLE REMOVAL: Removed Tefzel
cylindrical well; flushed well; refilled with buffer SEPARATION RUN
VOLTAGE: 3.33 kV; run for .about.90 sec; capillary cassette in
horizontal position CURRENT: 1.8 uA measured by hand-held
multimeter POLARITY: Negative at injection end; detector near
ground. BUFFER: Std Nucleophor .TM. buffer + 2.5 ug/ml EtBr
DETECTION: Auto scan from x = 190000 to x = 26000 via Cell Robotics
Smartstage; Capillary in focus +/-5 microns across scanned length.
Focus also intentionally misadjusted 1/2, 1, and 2 turns for scan
at 500 um/sec. scans. ______________________________________
FIG. 12 illustrates separation of the DNA fragments.
EXAMPLE 2
This example illustrates the separation of a series of
single-stranded oligonucleotides that differ by the addition of a
single base to the previous oligo. The sample consists of a
pdA.sub.10 fragment which was labeled with a fluorophore (FAM) on
the 5' end. The 5'FAM-pdA.sub.10 fragments were then enzymatically
extended with terminal transferase by addition of dATP onto the 3'
end of the fragments. This process gave a Gaussian distribution of
5'FAM-pdA.sub.x fragments, with X ranging from .about.20 to 75.
Separation of this sample mimics a DNA sequencing separation in
that single-stranded DNA fragments that differ by 1 base are
separated and detected by fluorescence detection.
______________________________________ Capillary: 10 cm length
.mu.m id window at 7.5 cm, internally coated with linear
polyacrylamide, positioned in separation plate illustrated in FIG.
1. Buffer: Solution of 10% .sup.w /.sub.v linear polyacrylamide in
1X TBE (89 mM Tris, 89 mM borate, 2 mM EDTA) plus 7M urea loaded
into the capillary via syringe. Anode/Cathode Resevoir Buffer: 1X
TBE, 7M urea Injection: 10 second at 2 kV electroinjection at
cathode end. Separation: 2 kV (.about.12 .mu.A current) const.
potential Sample: 50 nM total DNA; average of 1 nM each fragment
Detection: Static; fluorescence; 470-490 nm excitation > 520 nm
emission using detection scheme illustrated in FIG. 11, but with
static detection. Slit: 110 .mu.m .times. 20 .mu.m, positioned 7.5
cm from injection end. ______________________________________
The results are shown in FIG. 13.
EXAMPLE 3
This example illustrates the separation of some non-standard
samples of DNA. The samples were obtained by amplification of human
genomic DNA samples via the PCR (polymerase chain reaction)
process. Samples were not purified prior to use. Samples were
diluted with a predetermined concentration of a calibration
standard which contained PCR fragments of known sizes of 50, 100,
200, 300, 400, 500 . . . 1000 bp. The calibration standards were
obtained from Bio-Synthesis, Inc., Louisville, Tex. Samples were
diluted by 25% into the calibration standard and separated under
the following conditions:
______________________________________ Capillary: 9.2 cm length 30
.mu.m id 4% linear polyacryl- amide coated, window at 7.0 cm from
cathode end, positioned in an electrophoresis illustrated in FIG.
1. Buffer: Nucleophore .RTM. sieving buffer. (Dionex Corp.,
Sunnyvale, CA) + 2.5 .mu.g/mL ethidium bromide; loaded into
capillary via syringe. Cathode Buffer: 1X TBE (89 mM Tris, 89 mM
borate, 2 mM EDTA) + 2.5 .mu.g/mL ethidium bromide. Injection: 10
sec. at 3 kV electroinjection at cathode. Separation: 3 kV constant
potential. Detection: Excitation - 510-560 nm. Emission - >590
nm, using the static detection scheme illustrated in FIG. 11.
______________________________________
The expected size of this DNA sample was 211 bp. The size as
determined from the separation shown in FIG. 14 was 210 bp.
EXAMPLE 4
DNA Sequencing
A 96 multiple well plate with templates 1-8 to be sequenced placed
in all rows of respective lanes. Primers 1-8 are in all rows of
respective lanes, and all 4 bases are in all 96 positions.
The above array is sipped into the Base Plate. For sequencing, it
is desirable to simultaneously sip four samples and mix those
samples prior to sequencing. For example, with reactions involving
four separate reactions with color coded primers, place base Plate
with cover into instrument station which can control pressure
within the Base Plate, control temperature, and automatically add
reagents to the top of the Base Plate at the various positions.
Simultaneously add the polymerase to all 96 positions and start the
pressure/vacuum cycling to mix at the membranes and to start the
chain extension reaction.
With an eight-by pipetter, add fluorescently labeled, chain
extension terminators (for all 4 bases, color coded), to all 8
positions of the first row. Chain extension ends in these
positions, producing fragments extending from 10 (allowing for the
primer) to 120 bases, for all 8 templates.
After a predetermined period of time (matched to the polymerization
rate, controlled by buffer conditions), the same terminator mix is
added to the second row of eight lanes. Chain extension ends in the
second row, producing fragments extending from 80 to 220 bases from
the starting point, for all 8 templates. The stagger and overlap of
fragment sizes is determined by the time interval between
terminator additions, the concentration of polymerase and bases,
and the buffer conditions. The stagger of fragments may be obtained
by changing the relative concentration of terminators versus bases
at the various rows, or by starting all polymerization reactions
simultaneously and adding an enzyme-stopper at various time
points).
Additions of terminators, at predetermined time intervals is
continued for all rows.
The double stranded DNA is melted and injected and then separated
on the respective capillaries.
Each separation plate (at a given row) will have on-board sieving
buffer optimized for the fragment size range for that row which is
nominally a range of about 100 bases. Therefore reducing the
relative size resolution required to obtain single base
separation.
At the end of the entire process, one has simultaneously sequenced
8 templates, each for about 1200 bases, in about 30 minutes. Those
skilled in this also know how to re-prime and continue the
process.
The methods and apparatus of this invention have many advantages
for DNA sequencing. The primary advantages of this sequencing
method is that one obtains long read lengths by combining
continuous (or overlapping) read windows. Hence, within each row of
8 capillaries, one needs single base resolution for a defined read
window (e.g. from 490 to 610 bases). Therefore, the sieving buffer
for each row of 8 capillaries, on a given electrophoresis plate,
can be optimized for the particular read window.
Additional advantages are that the sequencing throughput is
increased by 3 to 10 fold over current methods and that small
volumes of template and reagents are required. The maine reason for
the higher throughput is that separation and reading is done
simultaneously for a large number of short capillaries.
Finally, though this sequencing method is ideally suited to the
devices of this invention, those experienced in the art of
sequencing will realize that this method may be practiced on other
devices such as standard gel-based DNA separation systems.
EXAMPLE 5
Method for Protein Binding Assay of Enkephalin Analogs
Capillary: 31 .mu.m ID fused silica, 10 cm mounted in a separation
plate shown in FIG. 1. Wash at beginning and end of day with
phosphate buffer and water (5 min).
Materials: Run buffer was 62.5 mM sodium phosphate, pH 8.5 with
0.01% (BSA) bovine serums albumin. Label (F-11,
YGGFLTSEK(-fluorescein)SQ (TANA Labs, Houston, Tex.), competitor
(YGGFLK- American Peptide Co., Sunnyvale, Calif.) and Fab'
monoclonal antibody fragment (Gramsch Labs Schwabhausen Germany)
were diluted in Run Buffer. Antibody and label were mixed at a
concentration of 12.5 nM each; this typically added to competitor
within 5 minutes after mixing. Competitor was diluted to several
concentrations. Add 40 .mu.l of Ab/label mixture to 10 .mu.l of
competitor and incubated 10 minutes before assaying. Mix by pumping
action of pipet. These mixtures were stable for several hours at
room temperature, if protected from evaporation and were held in
the dark. Final concentrations were: 10 nM Fab', 10 nM F-11, and 0,
20, 40 or 200 nM for the competitor.
Injection: The injection area of the separation plate was rinsed
with about 1 ml run buffer and all liquid removed. Using a
micropipet, 4 .mu.l of sample was delivered to the area between the
capillary end and (sample well) the electrode. Sample was
introduced into the capillary via electrokinetic injection for 10
seconds at 2.3 kV at the anode, at the end of which the injection
area was flushed with 4 drops of run buffer to remove residual
sample.
Separation: Voltage was set at 3 kV.
Detection: Laser light source; PMT; gain set at 10.sup.-4, 500
msec; detector window at 6 cm from injection. Argon ion laser, 488
nm excitation. Fluorescein was detected with a long pass filter
above 520 nm.
Analysis: Two areas in the fluorescent electropherogram measured as
shown in FIGS. 15A and B.
Area 1--under the free label peak.
Area 2--total fluoresence area, including the complex, free label,
and fluorescence between the two peaks due to label coming off the
Ab during migration.
Area 1/Area 2 is plotted vs. [competitor] as shown in FIG. 16.
EXAMPLE 6
______________________________________ Procedure for DNA Detection
Limit ______________________________________ Breadboard: separation
plate as shown in FIG. 1 Capillary: 10.1 cm of 30 .mu.m id fused
silica, covalently coated with polymeric layer of polyacrylamide;
window at 6.6 cm from cathrode end, and positioned in a separation
plate as illustrated in FIG. 1. Anode/Capillary Buffer: Nucleophor
.TM. sieving buffer (Dionex, Sunnyvale, CA) plus 2.5 .mu.g/ml
ethidium bromide. Cathode buffer: 1X TBE (89.5 mM Tris, 89.5 mM
borate, 2.0 mM EDTA, pH 8.3) plus 25 .mu.g/ml ethidium bromide.
Detector: as illustrated in FIG. 13, run in the static detection
mode. excitation 510-560 nm emission > 590 nm Injection Run: 30
sec at 3.0 kV @ cathode 3.0 kV constant voltage Sample: 0.033
.mu.g/ml Hae III digest of .O slashed.X 17 4 DNA (Sigma Chemical
Co., St. Louis, MO) in water
______________________________________
FIG. 17 shows that 12 picogram or 14 attomoles of 1353 bp DNA can
be detected and that 2-3 picograms or 14 attomoles of 310 bp DNA
can be detected.
EXAMPLE 7
FIG. 18 illustrates the efficiency of mixing very small volumes of
reagents as described in FIGS. 7a-e. Thus, 1.9 .mu.l of a dye
(xylene cyanole) was mixed with 1.9 .mu.l of water and the mixed
sample was passed back and forth through the membrane 80 for 0.4,
2.1 or 4.2 minutes.
The above examples are intended to illustrate the present invention
and not to limit it in spirit and scope.
EXAMPLE 8
This example demonstrates a combined sample sipping, sample
presentation, sample injection, separation, and scanning detection
of separated sample components.
______________________________________ Sipper/Mixer Sipper: 2.5 cm
lengths of 538 micron id fused silica capillary in a polycarbonate
plate Mixing 764 micron .times. 16 mm peek channels Block: in
polycarbonate block Top Plate: polycarbonate Membrane: 0.45 Micron
pore membrane Volume 7.30 microliters from array of PCR tubes,
Speed: each with 30 microliters sample Separator Breadboard: Plate
as shown in FIG. 9 Capillaries Type: 100 micron id fused silica
derivatized with 4% linear polyacrylamide coating Length: 109 mm
Window 90 mm Total: +/-45 mm from center Location: point of
capillaries Cleaning Flushed with water, then sieving buffer
Procedure: solution from syringe Samples: HAE III digest of pBR 322
DNA from Sigma Chemical Co. 10 microgram/ml in 0.5% TE buffer 30
microliters of sample were loaded into each of 6 PCR tubes as
primary sample array sample array Detector Optical Lamp: 75 watt
Xenon Iris: 1/8 opened N.D. Filters: ND1 and ND2 out Gain: 0.1
microamp/volt Time 50 Milliseconds Constant: PMT 850 volts Voltage:
Filter Set: G2A cube (ETBR):(Dichroic 580 nm, Exc. 510-560 nm, EM.
590 nm) Focus Focus on inner bore of capillary; Slits 150 .times.
20 & Slits: microns centered over inner bore Objective: 10x
Scanner: Cell robotics smartstage scanner Scan Speed: 1760
Microns/Sec X Scan X - 33000 TX x - .36000 microns Range: Buffers
Sample: 0.5X TE (5 mM TRIS, pH 7.5, 1 mM EDTA) Capillary: Stock
nucleophor buffer (Dionex Corporation) + 2.5 microgram/ml ethidium
bromide Common Stock nucleophor buffer (Dionex Resevoir:
Corporation) + 2.5 microgram/ml ethidium bromide Array 1 .times.
TBE (89.5 mM tris base, 89.5 mM Resevoirs: boric acid, 2 mM EDTA,
pH 8.3) + 2.5 microgram/ml ethidium bromide Power Bertan, modified
with timer/controller Supply: Polarity: Negative Injection Method:
Electrokinetic, from sampling plate on horizontal Duration: 15
seconds Voltage: 3.3 kV Sample Moved electrophoresis plate to
transfer Removal: capillaries from sample wells to run buffer wells
Separation Run Voltage: 3.3 kV Duration: 120 seconds Current: 156
microamps total for the 6 capillaries Detection: sequential scan of
the 6 individual capillaries at 1770 microns per second Data
PE/Nelson Model 1020 System: Input: 10 Volts fulls scale Sampling
20 Hertz Rate: ______________________________________
This simultaneously sipping an array of six samples, simultaneously
presenting the samples through the sample handling block,
simultaneously electroinjecting the presented samples into the
capillaries, simultaneously conducting the electrophoresis
following by analysis is demonstrated. FIGS. 19A-F illustrates the
separation and reproductability of separation of the system for DNA
sequences having a size between 72 and 310 base pairs.
* * * * *