Note: Descriptions are shown in the official language in which they were submitted.
CA 02407652 2002-10-28
Device and method for hybridizing double-stranded DNA samples to oligomer
arrays.
The invention concerns a device and a method for hybridizing double-stranded
DNA samples to
oligomer arrays.
Hybridization of sample DNA on oligomer chips, for example, oligonucleotide
arrays, is
conducted for detecting specific sequences in sample DNA. One possible
approach,
"sequencing by hybridization (SBH)p, in fact determines the complete sequence
of the sample
DNA or at least large portions thereof. Allele-specific hybridizations,
however, are also
conducted in order to detect specific changes in the sample DNA, e.g. point
mutations. The
sample DNA, however, usually is present in double-stranded form, since it has
previously been
amplified for the most part by means of PCR, and thus it is no longer
available essentially for
hybridizing with the oligomers. The present invention describes a device,
which serves for the
efficient hybridizing of double-stranded DNA to oligomer arrays.
A number of hybridizing chambers are known from the prior art. Thus, US-A-
5,100,775; US-A-
5,360,741; or US-A-5,466,603 describe hybridization chambers, which are
adapted to the most
varied objectives and requirements. Such hybridization chambers have in the
meantime
become available commercially in many forms, but generally they cannot be
separately
temperature-controlled. Hybridization chambers that are suitable for uptake of
specimen
carriers or slides are also known. In addition, there are foils, which are
self adhering at the
edges and which can form hybridization chambers by gluing onto the slide. A
pneumatically
controllable and temperature-controllable hybridization chamber is also known,
in which the
hybridization properties will be improved by movement of the hybridization
fluid.
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CA 02407652 2002-10-28
All of these devices, however, require that the double-stranded sample DNA is
either thermally
denatured or one strand is selectively separated prior to hybridization (e.g.,
a primer in PCR can
be labeled with biotin and one strand can be removed selectively from the
solution by binding to
streptavidin in the following step) or one strand is produced in excess by
enzymatic means, and
thus the segments hybridizing to the oligomer an-ay are not blocked by
complementary strands.
These methods represent not only an additional step, but they are also
expensive, particularly in
the case of biotin, and are often poorly reproducible in the case of enzymatic
reactions. If both
strands are to be detected, however, by the oligomer array, then only thermal
denaturing is
considered, without anything further. The problem, however, is so-called
reannealing, that is,
the complementary strands hybridize again with one another after the
denaturation, which can
occur more rapidly than hybridization with oligomers on the chip. This problem
is also not
solved by one-time denaturation. In contrast, if denaturation occurs in the
chamber, then DNA
fragments already hybridized to the oligomer array will again be removed.
The object of the present invention is thus to aeate a device, which overcomes
the
disadvantages of the prior art and makes possible an effective hybridization
of double-stranded
DNA samples.
The object is solved by the features of Gaim 1. Advantageous embodiments are
characterized
in the dependent sub-Gaims.
The object is thus solved by a device for hybridizing double-stranded DNA
samples to oligomer
arrays (oligomer chips), which comprises at least one pump transporting in two
directions, a
closed hybridization chamber, a cooling element and a heating element, whereby
the individual
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CA 02407652 2002-10-28
components are joined with one another in the above-named sequence, by
pathways for
transporting liquids.
The device described here makes possible a periodic denaturation of the DNA
sample, without
removal of DNA that has already hybridized to oligomers and thus overcomes the
problems
mentioned in the prior art. Due to the fact that the DNA sample is denatured
thermally prior to
introduction onto the chip and then is suddenly cooled, it is present in
single-stranded form for
the most part when it contacts the chip. Thus, a large part of the otherwise
double-stranded
DNA sample is available for hybridizing with the oligomer array. By pumping
the sample fluid
back and forth, the device assures that this process is repeated frequently
until a sufficient
portion of the double-stranded DNA sample has hybridized to the oligomers of
the chip. Also,
during the hybridization phase, a mixing occurs due to this process.
It is preferred according to the invention that the pump is a peristaltic
pump, a hose pump or a
piston pump. The pump will be able to transport small quantities of liquid
precisely in [both] the
aspiration and pressure directions. This can also be done according to the
invention by means
of valves, such as multiple-way valves, which are known to the person of
average skill in the art,
and these can be controlled andlor regulated in tum again externally.
It is particularly preferred that the pump is programmable or is controlled by
a computer. Such
controls and/or computer programs are known in and of themselves to the person
of average
skill in the art.
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CA 02407652 2002-10-28
Also, according to the invention, it is preferred that the hybridization
chamber comprises at least
one cover, with input/output channels passing through it and a tempering
block, with an
oligomer array that can be applied or fastened thereon.
it is particularly prefer-ed that a cooling unit is also present, on which the
tempering block is
arranged.
According to the invention, a device is also preferred, wherein the volume of
the hybridization
chamber amounts to less than 200 . I when the oligomer chip is inserted.
It is particularly advantageous that the hybridization chamber is equipped for
the uptake of
commercially available slides or microscope slides.
it is also preferred according to the invention that the cooling element
tightly surrounds the
pathway.
It is also preferred that the heating element tightly surrounds the pathway
and that the pathway
projects from the heating element via its open end. Alternatively, it is
preferred that the heating
element surrounds a sample vessel at least partially and that the pathway is
immersed by its
open end into the sample solution present in the sample vessel, and that this
pathway is
optionally conducted doom to the bottom of the sample vessel on the inside.
It is also preferred that the pathways are tubings and preferably comprised of
an inert material,
silicone rubbers, polytetrafluoroethylene, polyvinyl chloride, polyethylene
and/or special steel.
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CA 02407652 2002-10-28
Other inert materials are also considered and are known to the person with
average skill in the
art.
It is most particularly preferred that the hybridization chamber, the cooling
element, the heating
element and the tempering block can be temperature-controlled independent of
one another.
It is also preferred that the tempering block of the hybridization chamber is
simultaneously
configured as a cooling element.
It is also preferred that the volume of the pathways between the heating
element and the inlet
channel of the hybridization chamber is smaller than the volume of the
hybridization chamber
itself.
The present invention will be explained in more detail on the basis of the
attached illustrations.
Here:
Fig. 1a shows a schematic representation of a device according to the
invention in a first
example of embodiment,
Fig. 1 b shows a schematic representation of a device according to the
invention in a second
example of embodiment, and
Fig. 2 shows a perspective view of a form of embodiment of a hybridization
chamber according
to the invention.
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CA 02407652 2002-10-28
The subject of the present invention is a device for hybridizing double-
stranded DNA samples
on oligomer arrays (oiigomer chips) as schematized in Figure 1 alb. It is
comprised of a closed
hybridization chamber 1, 11 that can be temperature-controlled, a pump 4, 14,
a heating
element 3, 13 and a cooling element 2, 12, which are joined together each time
by pathways 5,
50, preferably plastic tubings, for transporting liquid.
Hybridization chamber 1, 11 (Figure 2) is preferably comprised of two parts, a
dish for uptake of
the oligomer array 23 and a cover 21, which preferably can be pressed together
by a hinge
mechanism. Preferably a recess is found in the cover for a sealing ring, which
forms the side
walls of the chamber. The cover also contains the ducts 22 for the tubing
connections 5, 15 or
other transport channels for liquids. The chamber can preferably be
temperature-controlled by
a Pettier element.
The pump preferably involves a tubing hose operating according to the
peristaltic principle or a
piston pump, which can be programmed for automatically conducting the method
by itself, or
can be controlled preferably by means of a PC. The sample is moved cyclically
by means of the
pump and is first denatured in the heating element, then cooled in the cooling
element and
subsequently hybridized in the hybridization chamber. After this, it is again
pumped into the
heating block and denatured. This process is cyclically repeated and the
device can preferably
conduct however many, but at least two, such cycles automatically, one after
the other.
The heating element as well as the cooling element are preferably comprised of
a metal block,
whose temperature is controlled most preferably by a Pettier element. !n a
preferred variant
both the heating element and the cooling element each surround a tubing,
through which the
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CA 02407652 2002-10-28
sample solution is transported. Alternatively, the heating element can take up
a vessel,
preferably comprised of plastic (e.g., "Eppendorf cup"). In this case, the
tubing or another
channel extends down to the bottom of this vessel in order to aspirate the
sample fluid therein.
The DNA sample can thus be transported to the cooling element and to the
hybridization
chamber. In another variant of the invention, a cooling element is not used,
but rather the
sample heated in the heating element is rapidly cooled by contact with the
temperature-
controlled hybridization chamber.
The hybridization chamber can take up oligomer chips, on which
oligonucleotides and/or PNA
oligomers (peptide nucleic acids) oligomer are immobilized. In a particular
prefer-ed variant, the
hybridization chamber takes up commercially available slides, such as are also
used in
microscopy. The volume which the hybridization chamber holds when the oligomer
chip is
inserted most preferably amounts to less than 200. I.
A subject of the present invention is also a method for hybridizing double-
stranded DNA
samples to oligomer arrays with the use of a device according to the
invention.
Another subject of the present invention is thus a device for hybridizing
double-stranded DNA
samples to oligomer arrays (oligomer chips), wherein a device according to the
invention as
described above is used and wherein the DNA sample is moved cyclically via the
pump and is
first denatured in the heating element, then cooled in the cooling element,
and subsequently
hybridized in the hybridization chamber, and is then again denatured in the
heating element,
whereby at least two such cycles are automatically conducted, one after the
other.
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Another subject of the present invention is a kit, comprising a device as
described above for
hybridizing double-stranded DNA samples to oligonucleotide arrays and one or
more oligomer
arrays or biochips and/or documentation for using the device and/or buffer
solutions for
conducting the hybridizations.
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CA 02407652 2002-10-28
Reference
list
1, 11 hybridization
chamber
2, 12 cooling element
3, 13 heating element
4, pump
14
5, 15 pathway
6 sample vessel
21 cover
22 inlet/outlet channels
23 oligomer array
24 tempering block
25 cooling unit
9
