Europaisches Patentamt

(19) European Patent Office

Office europeen
peen des brevets
brevets EP 0 715 715 B1

(12) E U R O P E A N PATENT S P E C I F I C A T I O N

(45) Date of publication and mention (51) Intel e G01N 2 7 / 4 4 7

of the grant of the patent:
27.01.1999 Bulletin 1999/04 (86) International application number:
PCT/US94/09633
(21) Application number: 94926595.3
(87) International publication number:
(22) Date of filing: 29.08.1994 WO 95/06243 (02.03.1995 Gazette 1995/10)

(54) GEL CASSETTE FOR ENHANCED ELECTROPHORETIC SEPARATION AND PROCESSES FOR
THE PREPARATION THEREOF
GELKASSETTE FUR EINE VERBESSERTE ELEKTROFORETISCHE TRENNUNG UND
VERFAHREN ZU DEREN HERSTELLUNG
CASSETTE DE GEL DESTINEE A LA SEPARATION ELECTROPHORETIQUE ET PROCEDES DE
PREPARATION ASSOCIES

(84) Designated Contracting States: • COLLIER, Charles, Forrest

AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL Wilmington, DE 19809 (US)
PT SE • ROBERTSON, Charles, William, Jr.
Centreville, DE 19807 (US)
(30) Priority: 27.08.1993 US 113480
(74) Representative: Jones, Alan John et al
(43) Date of publication of application: CARPMAELS & RANSFORD
12.06.1996 Bulletin 1996/24 43 Bloomsbury Square
London, WC1A2RA (GB)
(73) Proprietor: E.I. DU PONT DE NEMOURS AND
COMPANY (56) References cited:
Wilmington Delaware 19898 (US) FR-A- 2 677 894 US-A-5 149 417
US-A- 5 209 831 US-A- 5 234 559
(72) Inventors:
• BRUNK, Donald, Harvey
Wilmington, DE 19802 (US)

DO

LO
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give
notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in
o
a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art.
a.
99(1) European Patent Convention).
LU
Printed byJouve, 75001 PARIS(FR)
 1 EP 0 715 715 B1
Description
FIELD OF THE INVENTION
This invention relates to the field of gel electro-
phoresis. More particularly, this invention relates to ap-
paratus for separating molecular fragments across a ge-
latinous medium, and processes for the preparation
thereof.
BACKGROUND OF THE INVENTION
Gel electrophoresis is a standard tool in molecular
biology to separate molecular components, such as
DNA, RNA or protein, either for subsequent identifica-
tion or in a preparative procedure. In gel electrophore-
sis, the different mobility of ions under the influence of
an electric field (a function of electrical charge and/or
density) serves to separate molecular fragments as they
traverse the porous medium. It is common to then trans-
fer the separated fragments to a membrane that binds
the fragments permitting further processing directed to-
ward making an image ("blot") visible so that identifica-
tion can be accomplished.
Ordinarily this transfer is done with the fragments
of DNA or the like separated into bands distributed along
the body of the gel. The driving force is removed when
adequate separation has occurred. A membrane, usu-
ally a nylon, is placed on top of the gel so that the bands
transfer laterally to the membrane. This is known as a
Southern Blot and many variations are available.
An alternate procedure, direct blot, is described by
Pohl in U.S. Patents 4,631,120 and 4,631,122. There
the DNA fragments are run to the end of the gel and
transferred to a moving belt that is in contact with the
end of the gel. Pohl teaches concentration by angled
contact and programmed velocity. Pohl does not teach
the use of a prepackaged and disposable gel cassette
in conjunction with direct blot.
In U.S. Patent 5,234,559 incorporated by reference
herein, there is disclosed an improved direct blot proc-
ess and apparatus in which the separated fragments are
transferred from the end of the gel to a moving mem-
brane that is held on a frame. The framed membrane is
then subjected to the further manual or automatic
processing steps required to make an image visible for
identification such as the steps described in U.S. Pat-
ents 4,71 7,653 and 5,087,558 by Webster, Jr. which can
be followed by analysis such as that described by Hub-
ner in U.S. 4,885,697.
For convenience of handling and to insure accurate
alignment in use, the gel in the apparatus of that appli-
cation is held in a cassette which is cleaned after each
use and reused. It is one object of the instant invention
to avoid cleaning and reuse by providing a prepackaged
and disposable gel cassette. Prepackaged gels per se
are known, but none are in a cassette for direct blot.
It is an object of the instant invention to provide a
2
disposable gel cassette for direct blot procedures. It is
a further object to provide such a cassette in a system
in which the images obtained have bands which are well
separated and with enhanced concentration. To that end
5 the cassette of the instant invention provides an internal
angle near the contact to reduce the gel thickness and
concentrate the fragments. The apparatus of U.S. Pat-
ent 5,234,559 in which the instant gel cassette is useful
provides programmed velocity. Still further objects of the
10 invention are to provide a gel cassette which is conven-
iently filled in the gel casting process, easily packaged
for safe shipping, low cost, and convenient to insert into
and remove from the apparatus in the field.
15 SUMMARY OF THE INVENTION
There is disclosed herein a gel cassette in which
molecular fragments in a solution are separated elec-
trophoretically therein, comprising:
20
(a) a gelatinous substrate containing the molecular
fragments for separation at a first end thereof and
configured sufficient to concentrate similar separat-
ed molecular fragments at a second end thereof;
25 and
(b) containment means which supports the gelati-
nous substrate.
The gelatinous substrate (a) further comprises a top
30 surface to which the molecular fragments are added and
a bottom surface which contacts the containment
means and peripheral portions therebetween. The top
and bottom surfaces are essentially parallel to one an-
other in the region of the first end and the top and bottom
35 surfaces are essentially nonparallel (and preferably, ta-
pered) to one another in the region of the second end
so that the gelatinous substrate is narrower in cross sec-
tion between the top and bottom surfaces at the second
end than at the first end.
40 The containment means (b) further comprises a
base portion supporting the substrate (a) along its bot-
tom surface; a plurality of walls positioned essentially
perpendicularly to the bottom surface to support periph-
eral portions of the substrate (a); a top portion bearing
45 upon the top surface of the substrate; and a well portion
positioned relative to the top surface suitable for casting
integral wells within the substrate (a). These wells are
adapted to contain the solution including molecular frag-
ments for electrophoresis.
so There is also disclosed herein a process for the
preparation of a gel cassette for the electrophoretic sep-
aration of molecular fragments therein.
BRIEF DESCRIPTION OF THE DRAWINGS
55
Figure 1 is a perspective view, partly in cross-sec-
tion, of a gel cassette according to the invention.
Figure 2 is a cross-sectional view on the line 2-2 of
2
 3 EP 0 715 715 B1
Figure 1.
Figure 3 is a cross-sectional view similar to Figure
2 but shown with a closed lid.
Figure 4 is a side elevational view of a holding fix-
ture used for casting electrophoretic gel in the cassette
of the invention and for the shipment thereof.
Figure 5 is an end elevational view of the fixture of
Figure 4 on the line 5-5 of Figure 4.
Figure 6 is a perspective view, with an end eleva-
tional cross section, of a comb used with the cassette
of the invention.
Figure 7 is a perspective view of a preferred em-
bodiment of the lid of Figure 1.
Figure 8 is a cross-sectional view of the lid of Figure
7 taken on the line 8-8 and showing details of a locking
means.
Figure 9 is an enlarged cross section of the cassette
taken on the line 9-9 of Figure 2 showing details of the
gel cavity.
DETAILED DESCRIPTION OF THE INVENTION
The gel cassette 10 of the invention is best seen in
Figure 1. It comprises a base 12, and a cover portion
(lid 14) attached to base 12 by two hinge pivots 16. Lid
14 also acts as a handle to facilitate insertion of cassette
10 into processing apparatus. When lid 14 is in the open
position a locking means, such as a notch 18 on lid 14
mating with a pin 20 on base 12, holds the two members
in the open position. We prefer to use two such notch
18 and pin 20 arrangements, one on either side. We also
contour the end of hinge pivots 16 so that they act as a
bosses to bias the cassette into the proper position in
the apparatus in which it is used.
The lid 14 comprises a flat top 22 and two lid side
plates 24 which are contoured to receive hinge pivots
16 with holes 17 (not shown). Aslot26 forms an aperture
in flat top 22 for a purpose to be explained.
Reinforcing ribs 25 and 27 (not shown) stiffen lid 14.
Base 12 contains gel cavity 28 which is formed be-
tween a plurality of side walls 30 (only one of which is
seen in Figure 1), top wall 32, and bottom wall 34. Side
walls 30 are spaced inwardly from side plates 36 which
define the outer structure of base 12 and mount the hing-
es 16. The front of cavity 28 is closed by front wall 38
which is pierced by gel slot 40. The rear of gel cavity 28
is closed by rear wall 42 which is pierced by expanded
gel entry port 44 (as seen in Figure 2). The gel slot 40
and entry port 44 function to discharge electrophoreti-
cally separated components and to introduce gel to gel
cavity 28, respectively. Atop top wall 32 there is located
comb well 46, and top wall 32 within the confines of
comb well 46 is apertured by well guides 48 for entry of
the comb teeth 206 of the well-forming comb as will be
seen. We use thirteen well guides 48 to create in the
formed gel slab the desired number of lanes for electro-
phoresis. It should be noted that cavity 28 is tapered
down to slot 40 by taper 41 which serves to concentrate
4
the separated bands as electrophoresis is carried out
from the wells formed by the comb which is held in place
by the comb well 46 and the well guides 48 toward the
slot 40 where it is transferred to a membrane moving in
5 contact with front wall 38 upwardly (in use the cassette
is held substantially horizontal with the cassette 10 ori-
ented as shown in the figures).
Referto Figure 7. This embodiment is characterized
by adding comb support ribs 29 and 31 on the side op-
10 posite reinforcing ribs 25 and 27. These serve to support
comb 200 along the sides 202. See Figure 6.
Lid 14 preferably is configured so that when it is in
the open position, which is the operating position, (see
Figures 1 and 2) it is locked in place by pin 20 on base
is 12 being in notch 18. This is effected by having a locking
angle in the relationship between the two coacting parts.
To get the parts into this position requires that there be
some means to cam the structure of lid side plate 24
past pin 20 as lid 14 is opened. The way we do this is
20 shown in Figure 8. Formed on the extreme tip 23 of plate
24 are two quarter cones 33, 35 separated by a flat 37.
The apices of these cone structures are on flat 37 at
points 39, 41 . In operation, as lid 14 rotates about hinge
pivot 16 in hole 17, the beveled end of pin 20 contacts
25 quarter cone 35 and slides on its surface camming the
extreme tip 23 of lid side plate 24 away from side plate
36. As the motion continues it rides over flat 37 and into
notch 18. In the other direction of rotation of lid 14 there
is no camming provision and the pin 20 and notch 18
30 are locked. We have found that pin 20 will shear off if a
strenuous attempt is made to reclose lid 14. This is ben-
eficial because it prevents reuse of cassette 10 which
might cause dangerous cross contamination.
Now consider the configuration of the gel slab
35 formed in gel cavity 28. Resolution of the DNA patterns
produced by the direct blotting method can be enhanced
by appropriately shaping the gel. Ideally, the best reso-
lution results when all DNA fragments of a given size
are captured at the same location on the membrane. In
40 practice, this does not occur using a gel with a rectan-
gular cross section because similar DNA fragments are
distributed across the thickness of the gel, and are
spread across a distance equal to the thickness of the
gel when captured by the membrane. Furthermore, if the
45 speed of the membrane is slow, fragments of DNA which
can normally be identified as different from each other
by the traditional Southern blotting procedure may not
be resolved as separate because of pattern overlap.
The gel described herein has an internal shape
so which provides different path lengths for the DNA frag-
ments to allow similar DNA fragments to be captured in
close proximity to each other by a membrane positioned
perpendicular or nearly so to the major axis of the gel.
For purposes of this description, the gel is assumed
55 to be oriented horizontally (in the apparatus for which
cassette 10 is intended it is held within 2 degrees of hor-
izontal merely to provide drainage), and the membrane
vertically, with upward motion. The bottom surface of the
3
 5 EP 0 715 715 B1
gel is a horizontal plane. The upper surface of the gel is
parallel to the bottom for most of its expanse, but tapers
towards the bottom surface near the end which contacts
the membrane. This is best seen in Figure 9. After the
end of the taper, the upper surface parallels the bottom
surface for a short distance before reaching the end, the
thickness of the gel having been greatly reduced by the
taper. DNA fragments positioned near the bottom sur-
face of the gel have a straight path to the gel, whereas
fragments near the upper surface have a more convo-
luted, and thus longer, path. The speed and direction of
the membrane can be set such that DNAfragments hav-
ing a longer path length which exit from the gel at a later
time can be captured at the same point on the mem-
brane as the earlier DNAfragments, the membrane hav-
ing travelled in the interim so that the capture point co-
incides with the point at which the fragments exit from
the gel. It can now be understood that this shape can
be used along with an appropriate electric field and
membrane speed to achieve improved DNA pattern res-
olution. Although timing of the arrival of a DNA fragment
at the membrane is primarily related to the distance it
travels, there are less significant effects that should be
considered for a fuller understanding. The speed of trav-
el of a DNA fragment depends upon the strength of the
electric field through which it travels, and its mobility
which is a function of its size, shape, and the composi-
tion of the gel. As the cross section of the gel changes
in the region where it tapers, the electric field also
changes in intensity.
In use cassette is closed, as shown in Figure 3 and
4 and inserted into fixture 100 so that the openings into
gel cavity 28 are closed by rubber pads 104. Comb 200
(see Figure 6), which is fabricated from an elastomeric
material, is inserted into cassette 10 so that the inside
of slot 26 is contacted by wall 202, the inside of comb
well 46 is contacted by wall 204 and each of comb teeth
206 enters a comb guide 48. Teeth 206 are of a length
such that they penetrate part way into cavity 28. Thus
during the casting process they form a mold insert that
will create the wells in the gel slab that are to be charged
with innoculant. Each tooth 206 forms a leakproof seal
with its corresponding comb guide 48 so that gel does
not leak out during casting. Excess gel is not left in the
neighborhood of the well so that there are no particles
which might fall into the well and thus interfere with the
process.
Having reference to Figure 5, the needle of a sy-
ringe (not shown) which is loaded with gel forming liquid
(agarose for one) is inserted through a hole 105 in back
wall 108 of fixture 100 and passes through a similar hole
(not seen) in rubber pad 108 so that gel forming liquid
can be discharged into expanded gel entry port 44 and
cavity 28 filled. At least one hole 105 is needed, we pre-
fer two as shown. The second hole 105 may be used as
a vent. Slot 40 is closed by the other rubber pad 104
which is supported by front wall 106 of fixture 100. Thus
a slab of gel is formed completely bounded and having
6
a selected number of wells. Lip 102 acts as a convenient
handle for the combined cassette/fixture.
In use, a thin slab of agarose is cast in a four sided
slot. A comb penetrates the top side to form the wells.
5 The open end of the cavity holding the gel which is up-
stream relative to the direction of electrophoretic migra-
tion opens into a buffer chamber. The other open end,
which contacts the moving membrane, is tapered inter-
nally on the side away from the direction of motion of
10 the membrane near the contact end for band concen-
tration. The cassette itself is tapered externally away
from the direction of motion of the membrane for clear-
ance. A combination shipping cover/handle/lid is pivot-
ed near the upstream end of the cassette. The entire
is cassette is held in a clip for shipping which protects the
ends and bottom. Elastomeric pads at either internal
end of this clip seal both upstream and downstream ex-
posed ends of the gel during shipping and storage.
After casting, the gel is allowed to congeal and the
20 entire assembly is placed in a vapor impervious bag for
shipping and storage.
In the closed position the cassette is well protected
against physical damage. In use the clip (and a well-
forming comb) are removed first. The lid is pivoted to
25 the open position where a permanent (non-releasable)
snap lock is provided permitting the lid to serve as a han-
dle for insertion and removal of the cassette into and out
of a Separation and Transfer (S/T) module. The non-
releasable function of this lock prevents reuse of con-
so taminated gel. Bosses can be provided on the cassette
to releasably snap into the S/T structure to secure the
cassette in place and bias the cassette toward the fully
inserted position. In the instant apparatus this function
is provided by contours on the end of the hinge pivots.
35 The lid can be provided with a releasable snap lock in
the closed position. Additionally the functions of the clip
can easily be incorporated in the lid.
The consumer opens the bag, removes the assem-
bly of cassette 10 with comb 200 in place and fixture
40 100, removes fixture 100 and comb 200, opens lid 22,
and inserts cassette 10 into the automatic separation
and transfer apparatus designed for its use.
In such automatic apparatus as disclosed in U.S.
Patent 5,234,559 the surface upon which cassette 10
45 rests by design may be tilted below horizontal in the di-
rection of insertion to provide drainage. We use an angle
of 2 degrees. We prefer to remove this same 2 degrees
from the front wall 38 of cassette 10. We further prefer
to remove an additional 5 degrees from that wall (see
so angle "a" in Figure 9) so that the contact between the
transfer membrane which moves upwardly and the wall
38 takes place just before the end of the gel in slot 40
on surface 39.
The presence of a taper in cassette 10 shown by
55 angle "b" in Figure 9 allows the membrane to wrap
slightly around the end of the cassette and reduces the
vertical distance the cassette is in contact with the mem-
brane in the neighborhood of the gel, thus assuring good
4
 7 EP 0 715 715 B1
contact. The taper also mates with a similar taper in the
S/T module and in conjunction with the forward-biasing
action of the boss on the end of hinge pivots 16, locates
the cassette 10 in the apparatus precisely and securely.
Claims
1. A gel cassette (1 0) in which molecular fragments in
a solution are separated electrophoretically therein,
comprising:
(a) a gelatinous substrate for containing the
molecular fragments for separation at a first
end thereof (44) and configured sufficient to
concentrate similar separated molecular frag-
ments at a second end thereof (40); a top sur-
face (32) through which the molecular frag-
ments are added and a bottom surface (34)
which contacts a containment means and pe-
ripheral portions therebetween, said top and
bottom surfaces being essentially parallel to
one another in the region of said first end (42),
and said top and bottom surfaces being essen-
tially non-parallel to one another in the region
of said second end (40) so that said gelatinous
substrate is narrower in cross section between
said top and bottom surfaces at said second
end than at said first end, said top surface (32)
tapering toward said bottom surface (34) in the
region of said second end (40) such that said
top and bottom surfaces form a taper (41) in the
region of said second end; and
(b) a gel cavity (28) forming said containment
means which supports said gelatinous sub-
strate;
wherein said containment means includes
(i) a base portion (12) which supports said sub-
strate along said bottom surface thereof;
(ii) a plurality of walls (30) positioned essentially
perpendicularly to said bottom surface to sup-
port said substrate along said peripheral por-
tions thereof;
(iii) atop portion (32) which bears upon said top
surface of said substrate; and
(iv) a well portion positioned relative to said top
surface suitable for casting integral wells within
said substrate adapted to contain the solution
including molecular fragments for electro-
phoresis, said well portion comprising a comb
well (46) apertured at the bottom side thereof
by a plurality of well guides (48).
2. The gel cassette of Claim 1 wherein said walls (ii)
further include at least one aperture (44) there-
through in the region of the first end of said sub-
8
strate adapted for introduction of gel and an aper-
ture (40) therethrough in the region of the second
end of said substrate adapted for discharge of the
electrophoretically separated molecular fragments
5 to a suitable medium.
3. The gel cassette of either of Claims 1 and 2 wherein
said well portion (iv) further includes a comb (200)
having a plurality of teeth (206) which cast said in-
fo tegral wells when contacted by said gelatinous sub-
strate, said comb (200) having a wall (204) sealingly
contacting said comb well (46) and said teeth (206)
sealingly extending through said well guides (48).
is 4. The gel cassette of any one of the preceding Claims
wherein said containment means further includes a
cover portion (14) in hinged relation to said base
portion (12) and moveable from a first position
wherein said cover portion (14) extends over said
20 top portion (32) of said gel cassette, to a second
position wherein said cover portion (14) exposes
said top portion (32) sufficient to install the gel cas-
sette (10) in associated electrophoretic apparatus.
25 5. The gel cassette of Claim 4 wherein said base por-
tion (12) and said cover portion (14) lock into said
second position.
6. The gel cassette of any one of the preceding Claims
30 wherein a front well (39) is tapered toward said ge-
latinous substrate on both top and bottom sides of
said second end (40).
7. A process for the preparation of a gel cassette (1 0)
35 according to claim 1 comprising:
(a) providing containment means comprising a
base portion (12), a plurality of walls (30) posi-
tioned essentially perpendicularly to said base
40 portion, a top portion (32), and a well portion,
and wherein said walls further include at least
one aperture (44) therethrough in a first region
adapted for introduction of gel and an aperture
(40) therethrough in a second region adapted
45 for discharge of the electrophoretically separat-
ed molecular fragments to a suitable medium;
(b) positioning said containment means within
a fixture
(c) introducing gelatinous material into said
so containment means; and
(d) curing said gelatinous material;
characterised by:
55 said fixture having elastomeric material over-
laying said first and second regions; and
said gelatinous material is introduced by form-
ing a hole through said elastomeric material
5
 9 EP 0 715 715 B1
overlaying said first region and injecting gelat-
inous material therethrough.
Patentanspriiche
1. Gelkassette (10), in der Molekulbruchstucke in ei-
ner Losung elektrophoretisch getrennt werden, mit:
(a) einem gelatineartigen Substrat zur Aufnah- 10
me der Molekulbruchstucke fur die Trennung
an einem ersten Ende (42) des Substrats, das
hinreichend konfiguriert ist, urn ahnliche ge-
trennte Molekulbruchstucke an einem zweiten
Ende (40) des Substrats zu konzentrieren; ei- 15
ner oberen Flache (32), durch welche die Mo-
lekul bruchstucke zugegeben werden, und ei-
ner unteren Flache (34), welche mit einer Be-
haltereinrichtung und dazwischenliegenden
peripheren Abschnitten in Kontakt ist, wobei die 20
obere und die untere Flache im Bereich des er-
sten Endes (42) im wesentlichen parallel zuein-
ander sind, und wobei die obere und die untere
Flache im Bereich des zweiten Endes (40) im
wesentlichen nicht parallel zueinander sind, so 25 4.
dal3 das gelatineartige Substrat zwischen der
oberen und der unteren Flache am zweiten En-
de einen schmaleren Querschnitt aufweist als
am ersten Ende, wobei die obere Flache (32)
im Bereich des zweiten Endes (40) zur unteren 30
Flache (34) hin abgeschragt ist, so dal3 die obe-
re und die untere Flache im Bereich des zwei-
ten Endes eine Schrage (41) bilden: und
(b) einem Gelhohlraum (28). der die Behalter-
einrichtung bildet, die das gelatineartige Sub-
strat aufnimmt;
wobei die Behaltereinrichtung aufweist
(i) einen Basisabschnitt (12), der das Substrat
entlang seiner unteren Flache tragt;
(ii) mehrere Wande (30), die im wesentlichen
senkrecht zu der unteren Flache angeordnet
sind, urn das Substrat entlang seinen periphe-
ren Abschnitten aufzunehmen;
(iii) einen oberen Abschnitt (32), der auf der
oberen Flache des Substrats aufliegt; und
(iv) einen relativzu der oberen Flache angeord-
neten Vertiefungsabschnitt, der sich zum Gie-
I3en integrierter Vertiefungen innerhalb des
Substrats eignet, die so angepaBt sind, dal3 sie
die Losung aufnehmen, welche die Molekul-
bruchstucke fur die Elektrophorese aufweist,
wobei der Vertiefungsabschnitt eine Kammver-
10
tiefung (46) aufweist. die an ihrer Unterseite
durch mehrere Vertiefungsfuhrungen (48) mit
Offnungen versehen ist.
5 2. Gelkassettte nach Anspruch 1, wobei die Wande (ii)
ferner mindestens eine durchgehende Offnung (44)
im Bereich des ersten Endes des Substrats, die fur
den Eintrag von Gel angepaBt ist, und eine durch-
gehende Offnung (40) im Bereich des zweiten En-
des des Substrats aufweisen, die fur den Austrag
der elektrophoretisch getrennten Molekulbruch-
stucke in ein geeignetes Medium angepaBt ist.
3. Gelkassettte nach einem der Anspruche 1 oder 2,
wobei der Vertiefungsabschnitt (iv) ferner einen
Kamm (200) mit mehreren Zahnen (206) aufweist,
welche die integrierten Vertiefungen gieBen, wenn
sie mit dem gelatineartigen Substrat in Kontakt
kommen, wobei der Kamm (200) eine Wand (204)
aufweist, die abdichtend mit der Kammvertiefung
(46) in Kontakt kommt, und wobei sich die Zahne
(206) abdichtend durch die Vertiefungsfuhrungen
(48) erstrecken.
Gelkassettte nach einem der vorstehenden Anspru-
che, wobei die Behaltereinrichtung ferner einen
Deckel abschnitt (14) aufweist, der gelenkig mit
dem Basisabschnitt (12) verbunden und von einer
ersten Position, in welcher sich der Deckel ab-
schnitt (14) iiber den oberen Abschnitt (32) der Gel-
kassette erstreckt, bis zu einer zweiten Position be-
weglich ist, in welcher der Deckel abschnitt (14) den
oberen Abschnitt (32) hinreichend freigibt, urn die
Gelkassette (10) in dem dazugehorigen Elektro-
35 phoresegerat zu installieren.
5. Gelkassettte nach Anspruch 4, wobei der Basisab-
schnitt (12) und der Deckel abschnitt (14) in die
zweite Position einrasten.
40
6. Gelkassettte nach einem der vorstehenden Anspru-
che, wobei eine vordere Vertiefung (39) sowohl an
der Oberseite als auch an der Unterseite des zwei-
ten Endes (40) zu dem gelatineartigen Substrat hin
45 abgeschragt ist.
7. Verfahren zur Herstellung einer Gelkassette nach
Anspruch 1, mit den Schritten:
so (a) Bereitstellen einer Behaltereinrichtung mit
einem Basisabschnitt (12), mehreren Wanden
(30), die im wesentlichen senkrecht zu dem Ba-
sisabschnitt angeordnet sind, einem oberen
Abschnitt (32) und einem Vertiefungsabschnitt,
55 wobei die Wande ferner mindestens eine
durchgehende Offnung (44) in einem ersten
Bereich, der fur den Eintrag eines Gels ange-
pa!3t ist, und eine durchgehende Offnung (40)
6
 11 EP 0 715 715 B1
in einem zweiten Bereich aufweisen, der fur
das Ausbringen der elektrophoretisch getrenn-
ten Molekulbruchstucke in ein geeignetes Me-
dium angepaBt ist;
(b) Anordnen der Behaltereinrichtung innerhalb
einer Haltevorrichtung;
(c) Eintrag von gelatineartigem Material in die
Behaltereinrichtung; und
(d) Ausharten des gelatineartigen Materials;
dadurch gekennzeichnet, dal3:
die Haltevorrichtung elastomeres Material
aufweist. das den ersten und den zweiten
Bereich uberdeckt; und
das gelatineartige Material eingebracht
wird, indem ein Loch in dem elastomeren
Material angebracht wird, das den ersten
Bereich uberdeckt, und dadurch gelatine-
artiges Material injiziert wird.
Revendications
1. Une cassette de gel (10) a I'interieur de laquelle on
separe par electrophorese des fragments molecu-
laires dans une solution, comprenant:
(a) un substrat gelatineux pour contenir des
fragments moleculaires a separer a une pre-
miere extremite du substrat (44) et qui est con-
figure d'une facon suffisante a concentrer des
fragments moleculaires egalement separes a
une deuxieme extremite du substrat (40); une
surface superieure (32) a travers laquelle on
ajoute les fragments moleculaires et une surfa-
ce inferieure (34) qui vient en contact avec des
moyens de confinement, des parties peripheri-
ques etant situees entre les deux, lesdites sur-
faces superieure et inferieure etant sensible-
ment paralleles I'une a I'autre a proximite de la-
dite premiere extremite (42) et lesdites surfa-
ces superieure et inferieure etant sensiblement
non paralleles I'une a I'autre dans la zone de
ladite deuxieme extremite (40) de sorte que le
substrat gelatineux presente une section trans-
versale qui est plus etroite entre lesdites surfa-
ces superieure et inferieure a ladite deuxieme
extremite par rapport a celle a ladite premiere
extremite, ladite surface superieure (32) s'effi-
lant vers ladite surface inferieure (34) dans la
zone de ladite deuxieme extremite (40) de sorte
que lesdites surfaces superieure et inferieure
torment un effilement (41) dans la zone de la-
dite deuxieme extremite; et
12
(b) une cavite pour gel (28) formant lesdits
moyens de confinement supportant ledit subs-
trat gelatineux;
5 dans lequel lesdits moyens de confinement
comportent:
(i) une partie de base (12) supportant ledit
substrat le long de sa surface inferieure;
10 (ii) une pluralite de parois (30) positionnees
sensiblement perpendiculairement a ladite sur-
face inferieure pour supporter ledit substrat
long desdites parties peripheriques;
(iii) une partie superieure (32) qui s'appuie con-
15 tre ladite surface superieure dudit substrat et
(iv) une partie formant des puits qui est posi-
tionnee par rapport a ladite surface superieure
d'une facon permettant de couler des puits in-
tegres dans ledit substrat destines a contenir la
20 solution qui comporte des fragments molecu-
laires pour I'electrophorese, ladite partie for-
mant des puits comprenant un puits (46) a co-
quille, et dont la face inferieure est percee par
une pluralite de guides de puits (48).
25
2. La cassette de gel de la revendication 1, dans la-
quelle lesdites parois (ii) comprennent en outre au
moins une ouverture traversante (44) dans la zone
de la premiere extremite dudit substrat, destinee a
30 I'introduction du gel ainsi qu'une ouverture traver-
sante (44) dans la zone de la deuxieme extremite
dudit substrat, destinee a decharger des fragments
moleculaires separes par electrophorese sur un mi-
lieu adequat.
35
3. La cassette de gel selon la revendication 1 ou 2,
dans laquelle ladite partie a puits (iv) comprend en
outre une coquille (200) presentant une pluralite de
dents (206) qui realisent par moulage lesdits puits
40 integres lors du contact par ledit substrat gelati-
neux, ladite coquille (200) presentant une paroi
(204) qui vient en contact etanche avec ledit puits
(46) de coquille, lesdites dents (206) penetrant de
facon etanche dans lesdits guides-puits (48).
45
4. La cassette de gel selon I'une quelconque des re-
vendications precedentes, dans laquelle lesdits
moyens de confinement comprennent en outre une
partie de couvercle (14) associee de facon pivotan-
50 te avec ladite partie de base (12) et susceptible de
se deplacer d'une premiere position dans laquelle
ladite partie de couvercle (14) s'etend par dessus
ladite partie superieure (32) de ladite cassette de
gel, jusqu'a une deuxieme position dans laquelle la-
ss dite partie de couvercle (1 4) expose ladite partie su-
perieure (32) d'une facon suffisante pour I'installa-
tion de la cassette de gel (10) dans un appareil
d'electrophorese associe.
7
 13 EP 0 715 715 B1 14

5. La cassette de gel de la revendication 4, dans la-

quelle ladite partie de base (12) et ladite partie de
couvercle (1 4) se verrouillent dans ladite deuxieme
position.
5
6. La cassette de gel selon I'une quelconque des re-
vendications precedentes dans laquelle une paroi
frontale (39) s'effile vers ledit substrat gelatineux
des cotes superieur et inferieur de ladite deuxieme
extremite (40). 10

7. Un procede de preparation d'une cassette de gel

(10) selon la revendication 1, comprenant :

(a) la fourniture de moyens de confinement 15

comprenant une partie de base (12), une plu-
ralite de parois (30) positionnees sensiblement
perpendiculairement a ladite partie de base,
une partie superieure (32) et une partie de
puits, et dans lequel ladite paroi comprend en 20
outre au moins une ouverture traversante (44)
dans une premiere zone adaptee pour I'intro-
duction du gel et une ouverture traversante (40)
dans une deuxieme zone adaptee pour la de-
charge des fragments moleculaires separes 25
par electrophorese sur un milieu adequat;
(b) le positionnement desdits moyens de con-
finement a I'interieur d'une armature;
(c) I'introduction du produit gelatineux dans les-
dits moyens de confinement; et (d) le durcisse- 30
ment dudit produit gelatineux;

caracterise par le fait que:

ladite armature presente un materiau elasto- 35

mere en superposition sur ladite premiere et la-
dite deuxieme zones; et
on introduit le produit gelatineux en formant un
trou a travers ledit materiau elastomere recou-
vrant ladite premiere zone, et on injecte le pro- 40
duit gelatineux a travers ce trou.

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