|United States Patent
|Gorfinkel , et al.
||November 5, 2002 |
Method and apparatus for compression of DNA samples for DNA
A novel method of spatial compression of a DNA sample inside the capillary
for the gel capillary electrophoresis and an article for operating the method
are disclosed. In this method, after the electrokinetic injection, the sample is
compressed using the reverse electric field. A special DNA barrier material is
used to contain the sample in the capillary. We expect that this Electro Static
Compression (ESC) can increase the DNA concentration in the capillary by orders
of magnitude. In the proposed method and article the injection and compression
of the DNA sample are followed by subsequent electrophoretic separation in any
kind of sequencing container (for instance glass capillary). The use of the ESC
method will allow the increase of the length and the quality of the read, as
well as reduction of the DNA consumption. ESC method will also permit the use of
low power illumination sources including miniature and inexpensive light
emitting diodes (LED) instead of the Ar-ion laser for exciting fluorescent
labels in 4-color DNA sequencing.
||Gorfinkel; Vera (Stony Brook, NY);
Gouzman; Mikhail (Lake Grove, NY); Serge; Luryi (Old Field,
||Research Foundation of State University of
New York (Stony Brook, NY) |
||December 3, 1999|
|Current U.S. Class:
||204/451; 204/453 |
|Field of Search:
References Cited [Referenced
U.S. Patent Documents
|Foreign Patent Documents|
JAPIO abstract of Kanbara (JP40572178A).
Primary Examiner: Warden; Jill
Assistant Examiner: Noguerola; Alex
Attorney, Agent or
Firm: F. Chau & Associates LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims
priority to Provisional Application Serial No. 60/110,720 filed Dec. 3, 1998 and
incorporated herein by reference.
What is claimed is:
1. A method of spatial compression of a DNA
sample inside a capillary for gel capillary electrophoresis comprising the steps
providing a capillary having an inlet end and an outlet end;
providing a first electrode adjacent said capillary inlet end and a
second electrode adjacent said capillary outlet end;
injecting DNA sample into said inlet end of said capillary by applying a voltage
across said first electrode and said second electrode;
inlet end of the capillary with DNA barrier material;
DNA sample against the DNA barrier material by applying a voltage having a
polarity reversed to the electrokinetic injection voltage across said first
electrode and said second electrode; and
the compressed DNA sample by applying a voltage having a polarity reversed to
the compression voltage.
2. The method of claim 1, wherein the
compressed sample occupies about less than 40 .mu.m.
3. The method of
claim 1, wherein the DNA barrier comprises a high-density PAA gel including
about 30% T and 5% C.
4. The method of claim 3, further comprising the
step of limiting the compression by a space charge at an interface of the
aluminum foil, wherein the DNA sample does not adhere to the aluminum foil.
5. The method of claim 1, wherein the DNA barrier comprises an aluminum
foil disposed at a lower portion of the capillary.
6. The method of
claim 1, wherein an electrophoretically separate sample can be viewed under a
single photon detection module.
7. The method of claim 6, wherein the
single photon detection module comprises a dynamic range of about 23 bits.
8. The method of claim 6, wherein down to about 100 photons can be used
for identification of a fluorescent dye in the DNA sample.
9. The method
of claim 6, wherein the single photon module further comprises a four-color beam
wherein each color is modulated at a frequency range of about between 100 and
10. The method of claim 1 wherein the step of electrokinetically
injecting the DNA sample is performed at a voltage of about 16 kV and current of
about 10 .mu.A for about 180 seconds.
11. The method of claim 1 wherein
the step of electrokinetically injecting the DNA a sample is performed at a
voltage less than about 20 kV and current less than about 10 .mu.A.
The present invention relates to a method and
apparatus for compressing DNA samples for DNA sequencing.
The aim of the proposed method for in-capillary densification of the DNA
analyte is to obtain a very sharp compressed DNA samples which can be separated
in shorter separation times and experience less diffusion. Therefore, a need
exist for a new technique which has higher sensitivity and better resolution
than the conventional state-of-the-art techniques. This will moreover allow us
to extend the range of the separation to larger DNA fragments.
Notably, the new technique will be less expensive. Indeed, since in the
ESC method the injected DNA sample will be compressed into a narrow zone, the
injection time is not limited to rather short few seconds interval, but can be
extended to several minutes. This means that the method will allow to use
low-concentration DNA samples and minimize the waste of costly DNA material. The
intensity of the excited fluorescent radiation from the condensed DNA sample
will be strongly enhanced. This in turn will allow us to use low-intensity
illumination sources in the detection module of our DNA sequencing instrument
which will reduce the cost and expand the versatility of the sequencing
In the present section we provide a short review of the
state-of-the-art of densification techniques in the DNA capillary
electrophoresis (CE) of and discuss our motivation to develop a new method, for
the DNA sequencing industry.
BRIEF DESCRIPTION OF DRAWINGS
1 is depicts a three-tube electroinjection in accordance with the present
FIG. 2 is a schematic showing an automatic tube changer in
accordance with the present invention; and
FIG. 3 is a schematic diagram
of a programmable high voltage supply for use with the present invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
electrophoresis, a sample is usually introduced into the capillary by
electrokinetic injection. This method has the advantage of a built-in sample
compression mechanism. The mobility of injected DNA fragments is greatly reduced
when they enter the capillary, due to a steep gradient in the strength of the
electric fields at the inlet of the capillary [Wolf 95]. In gel CE, the mobility
is additionally reduced by a confining effect of the gel matrix. Because of this
deceleration, the crowding, or stacking, of the injected species occurs in the
capillary near its inlet. This stacking effect usually increases the sample
concentration of charged moieties by about an order of magnitude.
Unfortunately, the stacking phenomenon is greatly influenced by the
composition of the sample. DNA sequencing fragments are introduced into the
capillary along with other competing negative ions present in the sample
solution. The injection process is biased towards faster migrating small ions
and shorter DNA fragments. Due to its transient nature, the stacking process is
not amenable to quantitative evaluation. The effect of stacking under similar
conditions is often irreproducible because it depends on the local condition at
the injection point, such as the sample composition, solute chemistry, and the
spatial distribution of the applied electric field.
significant improvement in the stacking procedure was reported by B. Karger's
group at Barnett Institute [Salas-Solano 98, Ruiz-Martinez 98]. The researchers
instituted a meticulous sample clean-up procedure in which template DNA was
thoroughly removed and the total concentration of salts was drastically reduced.
In addition, they determined that the injection is more effective at an optimum
relatively low electric field of 25 V/cm. As the result of these changes, the
injected amount of the sequencing fragments was increased 10-50-fold. This
allowed the authors to increase the range of a reliable and reproducible
separation to above 1000 bp with a base calling accuracy of 99%.
remarkable result was achieved with a rigorous cleaning protocol that involved
an ultra-filtration through a special membrane treated with linear
polyacrylamide (for the template removal) and gel filtration for desalting. This
is an expensive and time-consuming process. Moreover, even this greatly
amplified stacking is effective only for small volumes of the injected sample.
The injection was conducted at the optimum electric field of 25 V/cm, which is
about one order of magnitude lower than that commonly used. The duration of
injection was optimized at 80 sec, about one order of magnitude longer than that
commonly used. Therefore, while the sample is concentrated to a higher degree,
the total injected amount remains close to that commonly used. The stacking
effect fades away as the length of the injected sample increases. The transient
deceleration force influences the motion of a charged fragment only momentarily
at the entrance of the capillary. When in the capillary, the fragment is
drifting under the injection field with a constant speed, according to its
molecular mobility. If the time of drift is long, the effect of stacking is
diluted and eventually lost.
Chien and Burgi [Chien 92] reported
stacking of very large injected volumes using their field-amplified sample
injection method for a free capillary electrophoresis. However, the method is
based on the electroosmotic effects and is unsuitable for gel electrophoresis.
To the best of our knowledge, no existing method offers inexpensive,
reliable and reproducible in-capillary densification of large volumes of DNA
samples. The intent of this proposal is to develop such a method.
Realization of the ESC method will lead to a significant improvement of
the gel CE analysis by reducing the cost of operation and equipment and
expanding the scope and versatility of CE. Below we present arguments to support
this point of view.
Minimizing Waste of the DNA Material in Capillary
In gel CE, the amount of injected material is delicately
balanced between two conflicting interests, resolution and detection. For better
detection, one needs larger amounts of material, but this leads to poorer
resolution. Indeed, for higher resolution it is desirable to have sample of
smaller length. This requirement arises for the following reason. During the
electrophoretic separation, DNA molecules aggregate in different zones according
to their size. These zones are detected at the output end of the capillary by
their fluorescence. Two zones become well resolvable when the separation between
their centers exceeds the length of the original injected sample. The separation
is proportional to the running time. However, at longer times the resolution is
degraded by diffusion processes that smear the peaks of the separated zones. In
this trade-off between the separation and diffusion, the optimum compromise is
usually found when the spacing between the zones is several hundred microns.
This sets the upper limit for the length of the injected sample such that its
volume is only on the order of few nanoliters. For a reliable detection, this
miniscule volume should contain a sufficient amount of DNA. This implies that
the injected sample material is highly concentrated. Such genetic material is
expensive and not always available. Sadly, the lion's share of it is routinely
wasted. As a practical example, in our standard run-of-the-mill separation a
sample is electrokinetically injected during 10 sec from a 2 .mu.L volume
containing the DNA sample (smaller volumes are difficult to handle). We estimate
that the injected portion of the DNA material does not exceed several percent
from the total amount DNA contained the source tube. If the run is successful,
the rest of the DNA sample is discarded.
With the development of the new
ESC technique that can compress large volumes of samples to a much smaller
volumes, this waste can be minimized. One can inject much larger volumes of
low-concentration sample material. The sample preparation routine can be
simplified and the cost of the material preparation greatly reduced. This is of
a great economic importance for the practical application of the DNA CE. As an
illustration, an average fully utilized sequencer working round the clock can
carry out twelve runs a day, or about four thousand runs per year. The cost of a
standard labeled template 100-run kit from ABI is about $600; the cost of the
material used per year is about $24,000, prohibitively high for an average
researcher. With our method, this cost will be dramatically reduced.
B.2.2. Cost Reduction of Sequencing Equipment
The ESC method
will produce much higher concentrations in the sample material. When the sample
is separated into zones, each zone will also be more concentrated. Since the
intensity of the excited fluorescence is directly related to the concentration,
we expect the fluorescence from the compressed zones to increase. The excess
fluorescent power of the compressed sample may be used not only to cut waste,
but also to improve the sequencing instrument and reduce its cost. Today a state
of the art sequencing instrument employs as an excitation source an Ar laser
with the output power of 30-40 mW and a price tag of several thousand dollars.
With the implementation of the ESC technique, a simple, lighter and cheaper 1 mW
laser will suffice. This will reduce the cost of the sequencing equipment. This
will also alleviate the bleaching problem that often plagues the CE analysis.
B.2.3. Significance of the ESC Method for Our Research
own research at SUNY SB, the employment of the ESC method will be a pivotal
point. We have developed a novel multicolor fluorescent detection technique for
the implementation in the DNA sequencing [Gorfinkel 1995]. The technique is
based on the illumination of the sample by several different light sources, each
with a different wavelength .lambda..sub.i and modulated in time in a
distinguishable way. Detection of fluorescence is then performed by a single
"color-blind" detector. This is conceptually different from the existing
multicolor detection techniques, where the distinct fluorophores are excited by
a single light source, but each kind of fluorophore is detected by a separate
detector. We have demonstrated that our technique of excitation yields a 10-fold
increase in the sensitivity compared with the standard technique.
have achieved a further dramatic increase in the sensitivity by utilizing a
single photon counter as detector of the fluorescent radiation, replacing the
standard analog photodetector. We have found that with the single photon
detection, the minimum number of photons necessary for a reliable identification
of a fluorescent dye can be as low as 100. We were able to reliably detect very
small photon fluxes down to 5.times.10.sup.-17 W. In the course of our research
we have developed a Single Photon Detection (SPD) module with an increased
dynamic range, 23 bits, which is a more than two order of magnitude improvement
compared to currently prevalent 16 bits formate.
Expanding the Range and
Versatility of the Gel CE Analysis.
The separation process is a constant
battle between the good and the evil--drift and diffusion. The drift process
with length-dependent mobility separates the zones and moves them further apart
while the diffusion smears the contrast between the separated zone peaks. In
this battle, time is against us because the separation distance is a linear
function of time while the diffusion length is sublinear in time. One of the
ways to minimize the diffusion relative to drift is to shorten the separation
time. However, the separation time is directly related to the linear size of the
original sample (to be detectable, the zones should be separated by a distance
larger than the original sample length). Therefore, for shorter separation times
the sample length should be reduced. With the ESC method, we can concentrate an
injected sample in a very small volume. We expect that this will dramatically
reduce the time required for the separation and, as a result, the extent of the
diffusion. With reduced diffusion, DNA fragments of the same molecular weight
can be resolved with shorter capillaries. Even more significantly, for the same
capillary length we can now resolve longer DNA fragments. This will further
expand the range of the separation process.
The method will give both
the researcher and the clinician an option to choose their own modus operandi.
Compression of larger volumes of low concentration DNA to standard
concentrations will reduce the cost of the PCR process as well as DNA waste.
Alternative regimes of compressing the samples of standard templates to a higher
concentration will reduce the required illumination. The sequencing apparatus
can be made lighter and significantly less expensive by using low-power
illumination sources. Sequencing regimes can be adjusted to particular research
or clinical needs.
The method will add to the DNA analysis a new
dimension--versatility. In combination with the novel method of fluorescence
detection (modulated multicolor illumination and single photon detection), the
method will allow the utilization of light-emitting diodes as the illumination
source. A light inexpensive (under $10K) CE analyzer for everyday commercial use
with LED-based detection could be put on the desk of every clinician. For very
special research-type high-resolution one-of-the-kind equipment, one could use
more expensive laser-equipped detection systems.
Description of the
Proposed Method and Apparatus
In this section, we shall outline the ESC
method for on-line densification of the DNA sample. First, we shall explain the
particulars of the method (Sect. D.1.). Then we shall report our preliminary
experiments with the reverse field compression with different barrier media
(Sect. D.2, D.3). These experiments were carried out on the experimental DNA
sequencing instrument designed and built at the SUNY SB Electrical Engineering
department as our research tool. In comparison with a commercial instrument, it
has a great advantage of flexibility: many experimental parameters such as the
length of the capillary, the times and the electric fields of various cycles,
etc. can be easily adjusted. In this section we give a brief description of our
instrument (Sect. D.4). At the end of this section, we describe our work plan
for the development of the compression method (Sect. D.5.).
D.1. How the
The sample is injected by the standard electrokinetic
injection with the notable difference that the length of the injected sample
(duration of the injection) is determined only by the concentration of the
sample in the cuvette and the desired concentration.
After the injection
stage, the process proceeds to the compression stage. The inlet of the capillary
is immersed into, or pressed against, a special barrier medium, impenetrable for
DNA fragments. The polarity of the electric field is reversed, so that the
negatively charged DNA fragments will be driven towards the inlet of the
capillary where they will be pressed against the barrier and accumulate there.
We believe the compression in our method will be more effective and
reproducible than that in the stacking method. The advantages are owing to the
fact that the compressing electric field created by an external power supply
will have a well defined value and can be applied during any desired time
interval. In contrast, the compressing electric field in the stacking method is
transient and uncontrollable. To begin the separation process, the inlet of the
capillary is immersed in the buffer cuvette and the applied field is reversed
The Use of Low Power Sources for Fluorescence Excitation
Because of high density of the DNA sample we can proceed with the
replacement of the costly multicolor laser based illumination source with an
inexpensive miniature LED source. The LED source module comprises a group of LED
devices with current drivers and programmable modulation circuits. The output
from the module is a four-color beam where each color is modulated at
frequencies in the range of 100-300 Hz. The fiberized LED illumination source
can be easily incorporated into our sequencing instrument described in the Sect.
D.2. Barrier Medium
Finding a medium that can use as a
block for the DNA molecules is the main goal of our proposed project. What
properties do we seek? Firstly, the medium should be a barrier for DNA
fragments. Next, it should either be a good conductor itself or be permeable for
small ions of the solute and water to provide a good conductance in the
A natural realization of the DNA barrier medium is a
very concentrated gel. The DNA mobility .mu. in gels has a very strong inverse
exponential dependence on the average pore size .alpha. [Noolandi 93], while the
mean pore size .alpha. varies inversely with the gel solid concentration C,
viz..alpha..varies. C.sup.-0.75 [Quesada 97]. It is possible to greatly suppress
the mobility by increasing the concentration of gel. For example, for
polyacrylamide gels, the average pore size was found to vary from 50 nm to less
than 1 nm when the solid concentration was increased from 3 to 30% [White 60].
On the other hand, even at their highest concentrations, these materials will
behave as reasonable conductors.
Another realization of the DNA barrier
is an electrostatic stopper. It can be realized as a fine metal grid positioned
at the inlet of the capillary and set at zero potential at the compression
cycle. Note that a barrier of the electrostatic type will block all charged
particles, independent on their size.
A solid metal electrode can also
be used as a barrier. However, such a barrier would not provide any carriers of
charge into the solution to compensate the charge of the DNA fragments
compressed at the barrier. This means that, in principle, the compression
process should be self-limiting. It will stop when the field of the DNA charge
screens the applied compressing field. The compression degree at this point
depends on the nature of the metal of the barrier and the distribution of
charges in the capillary. Our preliminary results (next section) with a solid
metal barrier suggest that this line of investigation is definitely worth
After the injection, the compression will be performed with
different blocking media, for some barriers, we may have to institute an
intermediate step between the compression and separation whose purpose will be
to dissociate the compressed material from the barrier interface. This step will
be carried out without moving the capillary from its position relative to the
barrier, by reversing the applied bias to the separation polarity.
the completion of the compression stage, the capillary will be immersed in the
standard buffer and the compressed sample will undergo the electrophoresis. The
compressed zones will be observed at the window with our fluorescence detection
system. We shall record the temporal length and intensity of the compressed zone
F and thus evaluate the size of the compressed zone d.sub.z and the relative
amount of injected DNA material. At this stage, we shall be using, as a test DNA
material, a set of genetic markers
D.3. Preliminary Experiments
D.3.1. Experimental Setup and Results
compression experiments were carried out in our capillary setup (Section D.4).
As an electrophoretic agent, we used a mixture of two dyes Xylene Cyanole and
Bromophenol Blue (Aldridge Catalog, items 33,594-0 and 11,439-1, respectively).
We determined that in a standard polymer separation medium, the electrophoretic
mobilities of the two dyes are close to those of DNA fragments with the
molecular lengths of about 80-100 bp and 12-20 bp, respectively. The dye mixture
has a bright blue color so that its dynamics in the tube can be observed with a
naked eye or through a microscope. The dye experiments produced a convincing
visual demonstration of the compression effect.
The dye mixture was
injected into the capillary at the injection voltage of 16 kV. The duration of
the injection was usually 3 min. The current during the injection was about 10
.mu.A. The length of the injected sample (light blue color) could be observed
visually through the stripped capillary walls at the inlet and was about 1 cm.
After the injection, the inlet of the capillary was blocked by the barrier and
the compression voltage of the same magnitude and duration was applied. After
the compression, the sample was examined by eye and under the microscope.
For the blocking medium we prepared a high-density PAA gel (T=30%,
C=5%). First, the capillary was inserted into the gel by piercing the gel with
the capillary. During the compressing cycle, the reverse current was of about
the same magnitude of 10 .mu.A. After the compression, we observed a blurred
blue stain at the end of the channel bored by the capillary in the gel. No
compressed material was observed in the capillary.
In the next
experiment, the end of the capillary was pressed against the solid surface of
the gel. Although the end of the capillary was rugged and the contact was not
tight, the current through the system still held at about 10 .mu.A. After the
compression, when the capillary was withdrawn, we observed an intensely bright
dark blue spot, similar to an ink drop, on the surface of the gel. No dye was
left in the capillary.
In still another experiment, we used aluminum
foil as a barrier. This time, the current decreased from 10 A at the beginning
of the compression down to zero value. The dye was compressed at the inlet of
the capillary to a cylinder less than 40 .mu.m in height (under the microscope
the height of the compressed dye was about half of the inner diameter of the
capillary). No dye was detected on the foil.
D.3.2. Interpretation of
The most plausible explanation of the first result is that the
dye escaped through defects and microcracks that were formed when the solid gel
was pierced by the capillary. We conclude that the immersion should be done
before the solidification. In case of PAA gels, the cross-linker should be added
after the immersion. Another approach is to use thermo-sensitive gels that
change their viscosity with temperature [Wu 98]. After the injection, the
immersion should be done when the gel is at its low viscosity state; for the
compression the gel should be brought to its high viscosity state.
second experiment demonstrated that the compression is a reality. The 1 cm long
sample was compressed into a virtually two-dimensional spot. In this
arrangement, however, the experiment did not work because the dye was left
deposited on the surface of the gel barrier. We suspect that it was due to a
rugged interface between the capillary and the barrier. Alternative explanation
is some binding between the compressed material and the gel. To improve the
interface, we intend to institute the protocol of capillary immersion into a
liquid gel and subsequent solidification. In addition, we should probably
introduce an intermediate step to pull the compressed material from the
interface into a capillary by applying a positive voltage for a short time while
the capillary is still immersed into a barrier medium.
Result of the
third experiment is encouraging. Note that the blocking barrier does not satisfy
the second condition of being permeable by the solvent ions. Indeed, not only is
the metal foil a barrier for DNA fragments, it is also a barrier for all
moieties present in the solution. Therefore, at the compressing polarity it
cannot supply a positive charge to compensate the negative charge of compressed
material. The compression is self-limiting due to the formation of a space
charge at the interface. The space charge formation plays a positive role,
because due to its presence the compressed material stays in the capillary and
does not adhere to the metal surface.
It is instructive to compare this
result with the standard stacking (10 sec injection) of the same dye where the
size of the injected zone is about 300 .mu.m. While the compressed zone was
almost an order of magnitude shorter (less than 40 .mu.m), the amount of
compressed material, however, was roughly 20 times greater (the injection time
in the compression experiment was 180 sec vs. 10 sec for the standard process).
Our preliminary dye compression results, though very encouraging, should
be taken with caution because they were obtained on the dye mixture whose
behavior under the ESC may be quite different from that of DNA. We intend to
conduct extensive experiments to verify the ESC technique with a series of
extensive experiments carried out on various genetic materials (see Section
Design and Development of a Programmable Module for Injecting DNA
Material into the Capillary
In the conventional capillary
electrophoresis, one has peculiar problem which arises during electroinjection
of the DNA material from a microcentrifuge "sample" tube into the capillary and
leads to a significant waste of precious material. The problem is that the
amount of material loaded into the sample tube is of necessity too large because
it must exist at a relatively high concentration. Lowering of the concentration
of labeled material in the tube would lead to longer electroinjection
time--which would be usually unacceptable because it would lengthen the initial
sample introduced in the capillary and degrade its subsequent separation and
sequencing. We have developed a proprietary technique for solving this problem,
as illustrated in FIG. 1. The three tubes in FIG. 1 illustrate three stages of
capillary electrophoresis (CE). The left and the right tube represent the
conventional loading and drive stages, respectively. However, the sample into
the capillary in the first stage has lower concentration of DNA fragments, takes
longer time to inject, and initially occupies longer stretch of the capillary,
compared to samples commonly used in DNA separation by CE. The low-concentration
sample is compressed by the negative drive in second stage. The technique is
expected to enhance the resolution of CE and at the same time lower the amount
of labeled material consumed. Referring to FIG. 1, three-tube electroinjection
is shown with the following features: (1) capillary; (2) microcentrifuge tube;
(3) labeled DNA sample; (4) "plug" gel of high electrical conductivity but
impenetrable for DNA fragments; (5) Buffer TBE/5M; (6) low-concentration DNA
sample, initially introduced in the capillary; (7) spatially "compressed" DNA
sample; (8) electrophoretic separation of "compressed" sample; (9) positive
"loading" voltage; (10) negative "compressing" voltage; (11) positive
"separating" voltage. Initially loaded sample (6) is too long to be used for
high-quality electrophoretic separation. It becomes adequate after the
The tube changing procedure will preferably be
automated. A design of the automatic apparatus is shown in FIG. 2. Referring to
FIG. 2. an automated tube changer is shown with the following features: (1)
capillary; (12) capillary fixture holder; (13) capillary fixture; (14)
replaceable tube; (15) reversible step motor; (16) drive for holder 12; (17)
electromagnet; (18) return motion spring; (19) conical guide with Pt electrode;
(110) cylindrical support; (111) rotating holder. The platinum electrode in (19)
connects to the high-voltage circuit with reversible polarity.
and Development of a Programmable Power Supply for Electrophoresis
preceding section illustrates the need for a controlled high-voltage power
supply (CHVPS), which would permit changing the magnitude and even polarity of
the applied voltage in a stable and programmable fashion.
The CHVPS will
be controlled automatically by the central control system of the apparatus but
the operator can assume manual control at any time. Referring to FIG. 3, a
programmable high voltage supply is shown for use with the present invention.
The block is assembled on the basis of the universal high voltage unit
(EMCO #4150). Control of the electric regime is provided by a built-in current
meter and a high-voltage gauge. The CHVPS will be controlled automatically by
the central control system of the apparatus but the operator can assume manual
control at any time.
Commonly assigned provisional applications U.S.
application Ser. No. 60/110,712 and 60/110,714 are incorporated herein by
G. LITERATURE CITED
[Chien 92]: Chien, R. L. and
Burgi, D. S., "Sample stacking of an extremely large injection volume in
high-performance capillary electrophoresis", Anal. Chem. 64, 1046-1050 (1992)
[Gorfinkel 95]: Gorfinkel, V. B. and Luryi, S., "Method and apparatus
for identifying fluorophores", U.S. Pat. No. 5,784,157 (filed 1995, issued July
[Noolandi 92]: Noolandi, J., "Theory of gel electrophoresis", in
Advances in Electrophoresis V5, A. Crambach, M. J. Dunn, B. J. Radola, Eds.
(1992) pp. 2-56 and references therein.
[Quesada 97]: Quesada, M. A.,
"Replaceable polymers in DNA sequencing by capillary electrophoresis", Current
Opinion in Biotechnology 8, 82-93 (1997).
Ruiz-Martinez, M. C. et al., "A sample purification method for rugged and
high-performance DNA sequencing by capillary electrophoresis using replaceable
polymer solutions; A. Development of the cleanup protocol", Anal. Chem. 70,
[Salas-Solano 98]: Salas-Solano, O. et al., "A sample
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Anal. Chem. 70, 1528-1535 (1998)
[White 60]: White, M. L. "The
permeability of an acrylamide polymer gels, J. Phys. Chem. 64, 1563-1565 (1960)
[Wolf 95]: Wolf, S. M. and Vouros, P. "Incorporation of the sample
stacking techniques into the capillary electrophoresis CF FAB mass-spectrometric
analysis of DNA adducts", Anal. Chem. 67, 891-900 (1995).
[Wu 98]: Wu,
C. et al. "Viscosity-adjustable block copolymers for DNA separation by capillary
electrophoresis", Electrophoresis, 19, 231-241 (1998).
preferred embodiments of a system and method of the invention (which are
intended to be illustrative and not limiting), it is noted that modifications
and variations can be made by persons skilled in the art in light of the above
teachings. It is therefore to be understood that changes may be made in the
particular embodiments of the invention disclosed which are within the scope and
spirit of the invention as outlined by the appended claims. Having thus
described the invention with the details and particularity required by the
patent laws, what is claimed and desired protected by Letters Patent is set
forth in the appended claims.
* * * * *