AU750086B2 - Electrophoresis method and apparatus for separating bio-organic molecules - Google Patents

Electrophoresis method and apparatus for separating bio-organic molecules Download PDF

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AU750086B2
AU750086B2 AU35030/01A AU3503001A AU750086B2 AU 750086 B2 AU750086 B2 AU 750086B2 AU 35030/01 A AU35030/01 A AU 35030/01A AU 3503001 A AU3503001 A AU 3503001A AU 750086 B2 AU750086 B2 AU 750086B2
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teeth
loading
zone
plate
separation
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David Clark
Johann Goudberg
Barton G. Selby
Binh Vo
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Applied Biosystems Inc
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PE Corp
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AUSTRALIA
Patents Act COMPLETE SPECIFICATION
(ORIGINAL)
Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: PE Corporation (NY) Actual Inventor(s): Barton G Selby, Johann Goudberg, Binh Vo, David Clark Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: ELECTROPHORESIS METHOD AND APPARATUS FOR SEPARATING BIO-ORGANIC
MOLECULES
Our Ref: 640665 POF Code: 259463/455387 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1la- ELECTROPHORESIS METHOD AND APPARATUS FOR SEPARATING BIO-ORGANIC MOLECULES The present application is a divisional application from Australian Patent s Application number 35656/99, the entire disclosure of which is incorporated herein by reference.
Field of the Invention The present invention relates to the electrophoretic separation of analytes of interest, such as bio-organic molecules. In particular, the invention provides an improved slab gel electrophoresis apparatus, and method of use, for electrophoresis of samples using ultra-thin gels.
Background of the Invention Electrophoresis is a popular method of separating large molecules, such as DNA fragments or proteins, from a mixture of similar molecules. Generally, the method involves passing an electric current through a porous medium containing the mixture.
Each kind of molecule travels through the medium at a different rate, depending on its electrical charge and size. This movement produces a series of bands in the medium.
Each band corresponds to a molecule or fragment of a particular size, progressively decreasing down the gel. Agarose and acrylamide gels are media commonly used for electrophoresis of proteins and nucleic acids.
Due in large part to its usefulness in nucleic acid sequencing, slab gel electrophoresis has become increasingly important in the field of biotechnology. A slab gel apparatus is typically prepared by disposing two glass plates face-to-face, with spacers interposed at both vertical edges to establish a gap therebetween. In one approach, the plates re clamped together along the vertical edges and a seal is placed along the top or bottom edge. The gap between the plates is then filled with gel solution, and the gel is allowed to set. Next, the top and bottom of the polymerized gel are placed in contact with respective buffer reservoirs. Samples are loaded into sample loading slots at one end of the gel, and an electric field is set up across the gel. The electric field causes the molecules to move from the loading slots into the gel and to separate according to size.
Slab gels of very thin cross-section, in the direction perpendicular to the plane of electrophoresis, are particularly popular for nucleic acid sequencing thanks to a number of advantages they offer over thicker gels. For example, ultra-thin slab gels 0.1 mm to 0.3 mm thick) can be electrophoresed at a higher voltage than traditional, thicker gels because of more efficient heat dissipation, thereby permitting a faster electrophoretic run.
Ultra-thin gels also offer the advantage of higher resolution, because less sample is W:\liona\NKSpccis\Div of 35656,doc WO 99/54721 PCT/US99/0835 required and there is less time for diffusion during electrophoresis. Additionally, owing to their thinness, ultra-thin gels can be fixed for autoradiography with relative quickness and ease.
In using ultra-thin gels, it is desirable to increase lane density to achieve higher sample throughput. The dimensions of the sample lanes in the separation medium will typically depend on the dimensions of the regions into which the samples are loaded, the spacing between sample loading regions, the duration of time needed to transport the samples into the separation medium, and the level of diffusion during electrophoresis, for example.
Two types of sample-holding slots, known as "wells," are generally used in slab gels. In one approach, the wells are molded directly into the gel material using a template.
The template, known as a comb, comprises a plurality of square-ended, evenly spaced protrusions extending off one edge of an elongate support member. To form the wells, the @e e S protrusions of the comb are inserted into the top of a gel solution before it has polymerized.
The gel is then allowed to solidify. After the template is removed from the polymerized gel, empty wells remain for receiving samples. In such a construction, the loaded samples are separated from each other by finger-like extensions of the polymeiized gel. In the other common type of well, often used with ultra-thin gels, sample-holding slots are created by inserting narrow, wedge-shaped, pointed protrusions of a "shark's tooth" comb partially 20 into the top of the gel after it has polymerized. Samples are loaded into empty spaces bounded by adjacent teeth of the comb and the inner surfaces of the glass plates. The teeth of the shark's tooth comb, thus, serve as barriers to prevent the mixing of adjacent samples.
In most slab gel setups, the thickness of each well is determined by the thickness of the gel. Consequently, ultra-thin gels typically have ultra-thin sample wells. As a practical matter, it is difficult to load gels having wells that have a thickness of 0.2 mm or less. In this regard, sample loading is usually attempted by inserting a loader tube, such as a flat pipette tip or tapered glass capillary, having an extremely small outer diameter, between the two plates to deposit samples into the wells. To prevent sample mixing, it is desirable to inject the sample as close to the gel as possible. Under circumstances where the wells are very thin and the lane density is high, however, it can be very difficult to maneuver the tip of the loading implement between the plates and into position proximate the gel.
Moreover, the loader tubes themselves can present additional problems. For example, viscous samples can be difficult to deposit with such small loading devices because their WO 99/54721 PCT/US99/0835 i narrow bores easily clog and the tapered tips readily break. Also, loader tubes having ultra-small tips can be expensive and difficult to obtain.
As previously discussed, two types of templates, or combs, haive traditionally been used for forming sample-loading slots; namely, square-tooth combs and shark's tooth combs. Although satisfactory in certain respects, each type of comb nevertheless presents certain problems.
Often, the bottom of a well formed with a square-tooth comb will have an uneven surface. This can be caused, for example, by unpolymerized gel material remaining in the well and/or by tearing of the gel upon withdrawal of the comb. An uneven gel surface, in turn, can result in crooked bands and/or skewed lanes during electrophoresis.
The projections, or teeth, of a shark's tooth comb tend to be very thin and flexible.
This is especially true of combs having a large number of teeth 48 teeth or higher).
Upon pressing a comb into a polymerized gel, the teeth encounter a slight amount of resistance. Unfortunately, thin teeth sometimes bend as a result of such resistance. Bent teeth can distort the gel, resulting in crooked bands and/or skewed lanes during electrophoresis. Bent teeth can also change the volume between adjacent teeth for accommodating sample. Thus, maximum sample volumes can vary from well to well.
S"Moreover, thin, flexible teeth have also been associated with cross-leakage of samples.
Thin teeth also tend to be weak and prone to breakage.
20 Generally, the teeth of shark's tooth combs are especially thin towards their outer ends, in the region whereat the interface of the well and the gel is defined. For this reason, the barrier between adjacent wells is often not very substantial. Upon electrophoresis, the lanes run very close to one another. That is, there is insufficient separation, or "dark space", between lanes. As a result, it is sometimes difficult to distinguish the border between adjacent lanes. This can cause misreads by automated sequencing equipment.
For example, the reader might interpret two adjacent bands, representing molecules of a particular length or molecular weight, as a single band. Consequently, the reader would report only one band, which it would assign to only one of the two adjacent lanes. Thus, the closely spaced band in the adjacent lane would go unreported. Of course, lane drifting can further complicate this problem, especially where lanes actually converge into one another.
In an effort to accommodate a large number of wells on a single gel, some shark's tooth comb designs have the teeth spaced very close together. With such combs, the apertures between adjacent teeth do not provide much space for accommodating sample.
3 -4- This is particularly true with combs designed for conventional ultra-thin slab gel setups. So, the user is restricted to using only very small sample volumes less than 1 microliter).
The widespread use of 96-well trays in the field of biotechnology makes the use of a 96-well, slab gel electrophoresis apparatus especially attractive. For example, a single sequencing run could match the contents of a 96-well thermocycler plate. The known slab gel setups, however, are not able to complement 96-well trays in this manner. For practical purposes, the wells formed by conventional, square-ended combs are too large to fit 96 of them on a single slab gel. Regarding shark's tooth combs, problems associated with thin teeth, such as those discussed above, render these inadequate for creating 96 wells on a single slab gel.
In view of the foregoing, the need is apparent for a method and apparatus that realize the advantages offered by ultra-thin slab gel electrophoresis having 15 high-lane density, while avoiding the deficiencies and problems associated with the prior setups.
•The discussion of the background to the invention herein is included to o explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common 20 general knowledge in Australia as at the priority date of any of the claims.
Throughout the description and claims of the specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.
Summary of the Invention In one aspect the present invention provides a slab gel electrophoresis apparatus having a pair of spaced, confronting plates defining a multi-lane separation zone adapted to hold a separation medium and an elongate upper loading zone, and a separation medium held within said separation zone; further comprising: a comb having an elongate support member and a series of teeth extending from one edge of said support member; wherein each tooth has a first tapered portion which tapers gradually beginning proximate to said support member and extending along at least half of W:\fiona\NKI\Spcies\div of 35656.do.
4a the tooth's longitudinal axis, and (ii) a second tapered portion, more sharply tapered than sais first tapered portion, which begins immediately beyond said first tapered portion and extends along the tooth's longitudinal axis to an end of the tooth distal from said support member; wherein said teeth are positioned between said plates in said loading zone with the distal end of each tooth penetrating said separation medium, thereby forming a plurality of elongate loading regions, between adjacent teeth, that communicate with the separation zone.
In one embodiment, the separation medium held between the confronting plates extends from the separation zone at least partially into the loading zone.
The elongate recess preferably spans at least two adjacent loading regions formed by the comb. More preferably, the elongate recess spans at least 48 adjacent loading regions, and more preferably, spans at least 96 adjacent loading regions. In another W:\AfionNK\Spies\div of 35656.doc WO 99/54721 PCT/U S99/0835 I embodiment, the elongate recess extends across at least 70%. and preferably at least of the lateral width of one of the plates.
In a further embodiment, the plate-to-plate distance within the separation zone is less than 0.3 mm, and the plate-to-plate distance within the loading zone is greater than 0.3 mm. In a related embodiment, the plate-to-plate distance within the separation zone is about 0.2 mm. In another related embodiment, the plate-to-plate distance within the loading zone is from 1.5 to 3 times the plate-to-plate distance within the separation zone.
In another embodiment, the teeth of the comb extend off one edge of an elongate support member. In one embodiment, each tooth contains a first region beginning proximal to and extending away from the support member, such that the first region is optionally tapered, and (ii) a terminal region that is more tapered than the first region and has a pointed end, for forming a liquid-tight seal with the top of the separation medium. In a more specific embodiment, each tooth has a first tapered portion which tapers S. gradually beginning proximate to the support member and extending along most of the tooth's longitudinal axis, and (ii) a second tapered portion, more sharply tapered than the first tapered portion, which optionally begins immediately beyond the first tapered portion and extends along the tooth's longitudinal axis to an end of the tooth distal from the support member.
*too In a related embodiment, the teeth of the comb extend off one edge of an elongate I" 20 support member and are positioned between the confronting plates in the loading zone with their distal end regions penetrating a surface of the separation medium. In this embodiment, the spacing between edges of adjacent teeth at their distal end regions where the teeth cross the surface of the separation medium is a multiple less than less than 3 times the width of the individual teeth where the teeth cross the surface of the separation medium.
Another aspect of the invention provides a comb for a slab gel electrophoresis apparatus having a pair of spaced, confronting plates defining a multi-lane separation zone adapted to hold a separation medium and an elongate upper loading zone, and a separation medium held within the separation zone. The comb includes an elongate support member and a series of teeth extending from one edge of the support member. The teeth are positioned between the confronting plates in the loading zone with their distal end regions penetrating a surface of the separation medium. The teeth and confronting plates define a plurality of loading regions, between adjacent teeth, that communicate with the separation zone. The spacing between adjacent teeth at their distal end regions where the teeth cross WO 99/54721 WO 9954721PCTIUS99/0835 I the surface of the separation medium is a multiple less than 3 of the width of the individual teeth.
In one embodiment, the spacing between edges of adjacent teeth at their distal end regions where the teeth cross the surface of the separation medium is less than 1.35 num, and the width of each individual tooth where the teeth cross the surface of the separation medium is greater than 0.45 nun. In a related embodiment, the spacing between edges of adjacent teeth at their distal end regions where the teeth cross the surface of the separation medium is between 1.2 1.3 mm, and the width of each individual tooth where the teeth cross the surface of the separation medium is between 0.5 0.6 mmn.
In-another embodiment relating, at least one of the plates has an elongate recess across an upper portion thereof. The recess forms, together with the confronting plate, an elongate upper loading zone having a plate-to-plate distance that is substantially constant throughout the upper loading zone and (ii) is greater than the plate-to-plate distance within the separation zone. The plate further includes an elongate tapered portion joining the separation zone with the loading zone. The elongate tapered portion has a plate-to-plate distance that progressively decreases from the loading zone to the separation zone. Further in this embodiment, the separation medium extends al least partially into the *oeo tapered portion.
A further aspect of the invention provides an improvement for a slab gel 000 20 electrophoresis apparatus having a pair of spaced, confronting plates defining a multi-lane 0 000. separation zone adapted to hold a separation medium and an elongate upper loading zone, .*0.and a separation medium held within the separation zone. The improvement includes a ocomb having an elongate support member and a series of teeth extending from one edge of the support member. In one embodiment, each tooth contains a first portion beginning proximal to and extending away from the support member, such that the first region is optionally tapered, and (ii) a terminal portion that is more tapered than the first portion and has a pointed end, for forming a liquid-tight seal with the top of the separation medium. In a more specific embodiment, each tooth has a first tapered portion which tapers gradually beginning proximate to the support member and extending along most of the tooth's longitudinal axis, and (ii) a second tapered portion, more sharply tapered than the first tapered portion, which optionally begins immediately beyond the first tapered portion and extends along the tooth's longitudinal axis to an end of the tooth distal from the support member. The teeth are positioned between the plates in the loading zone with the distal end of each tooth penetrating the separation medium, thereby forming a plurality of WO 99/54721 PCT/US99/0835 I elongate loading regions, between adjacent teeth, that communicate with the separation zone.
In one embodiment, the teeth have front and rear surfaces that are substantially planar and parallel to one another.
In another embodiment, the separation medium within the separation zone is less than 0.3 mm thick, and the teeth of the comb are greater than 0.3 mm thick. In a related embodiment, the face-to-face thickness of the comb is 1.5 3 times greater than the plateto-plate distance within the separation zone.
In a further embodiment, the comb contains at least 97 teeth for forming at least 96 sample loading regions.
In another embodiment, each loading region has substantially the shape of an inverted In an additional embodiment, at least one of the plates has an elongate recess oo formed in the upper portion thereof. The recess forms, together with the confronting plate, S 15 an elongate upper loading zone having a plate-to-plate distance which is substantially constant throughout the upper loading zone and (ii) is greater than the plate-to-plate •distance within the separation zone. The plate further includes an elongate tapered portion joining the separation zone with the loading zone. The elongate tapered portion has a plate-to-plate distance that progressively decreases from the loading zone to the separation zone. Further, the separation medium extends at least partially into the tapered portion.
Another aspect of the invention provides a comb for a slab gel electrophoresis apparatus having a pair of spaced, confronting plates defining a multiilane separation zone adapted to hold a separation medium and an elongate upper loading zone, and a separation medium held within the separation zone. The comb includes an elongate support member and a series of teeth extending from one edge of the support member. The teeth are positioned between the confronting plates in the loading zone with their distal end regions penetrating a surface of the separation medium. The teeth and confronting plates define a plurality of loading regions, between adjacent teeth, that communicate with the separation zone. The teeth have front and rear surfaces that are substantially planar and parallel to one another. Further, the face-to-face thickness of the comb is a multiple of greater than 1.1, and preferably greater than 1.5, and more preferably greater than 2, times the plate-toplate distance in the separation zone.
An additional aspect of the invention provides a method of preparing a slab gel electrophoresis apparatus for use in separating bio-organic molecules. The method WO 99/54721 PCT/US99/08351 includes the step of providing a pair of spaced. confronting plates defining a multi-lane separation zone and an upper loading zone, a separation medium held within the separation zone, and a comb having a plurality of teeth adapted to be inserted into the loading zone.
The method also includes the step of inserting the comb into the loading zone so that distal ends of the teeth penetrate an upper portion of the separation medium, thereby forming a plurality of loading regions, between adjacent teeth, that communicate with the separation zone. In a preferred embodiment, the loading regions are of substantially rectangular longitudinal cross-section, and the plate-to-plate distance within the loading regions is greater than the plate-to-plate distance in the separation zone.
These and other features and advantages of the present invention will become clear from the following description.
Brief Description of the Drawings objctThe structure and manner of operation of the invention, together with the further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which identical reference numerals identify similar elements, and in which: Figure 1 is a perspective view of a slab gel electrophoresis apparatus, showing a pair of confronting plates separated by a pair of spacer strips and a comb poised above the plates, according to an embodiment of the present invention.
Figure 2 is a side cross-sectional view taken along the line 2-2 of Figure 1.
Figure 3 is a front elevational view showing the slab gel electrophoresis apparatus of Figure 1 with the comb inserted into the loading region between the confronting plates.
Figure 4 is a side cross-sectional view taken along the line 4-4 of Figure 3.
Figure 5 is a front elevational view showing a comb according to an embodiment of the present invention.
Figure 6 is a side elevational view of the comb of Figure Figure 7 is a partial enlarged view showing two teeth of the comb of Figure Detailed Description of the Invention One aspect of the present invention provides an improved apparatus for the electrophoretic separation of bio-organic molecules, particularly nucleic acids such as DNA, using an ultra-thin gel.
WO 99/54721 PCT/US99/08351 Referring in detail to the drawings. Figure 1 is a perspective view of a slab gel electrophoresis apparatus, indicated generally by the reference numeral 12, constructed in accordance with the present invention. A pair of plates 14, 16 are disposed face-to-face with a pair of spacer strips 18, 20 interposed at both vertical edges to establish a gap 22 between the plates. The plates 14, 16 are compressed together by any suitable means (not shown), which could include any of various types of clamps or clips along their lateral edges. A comb 24 is shown poised above the plates 14, 16 prior to insertion into the upper region of the gap 22.
An electrophoresis medium layer, such as 23 shown in Figure 2. is interposed between the two plates 14, 16 and occupies most of the gap 22. The electrophoresis medium layer 23 may be of any suitable type. For example, the electrophoresis medium layer 23 may comprise an agarose or acrylamide gel, optionally covalently crosslinked, and optionally containing a suitable denaturant such as urea. In a preferred embodiment, the electrophoresis medium layer 23 is formed of a 5% polyacrylamide gel.
15 Referring again to Figure 1, the spacer strips 18, 20 establish the width of the gap 22 between the two plates 14, 16 and, hence, the width for the interposed gel. The spacer strips 18, 20 are formed of a non-conductive material capable of forming a watertight seal, such as polyester or MYLAR® (DuPont). The gap 22 defines a space that serves as both a mold cavity for the gel and the space to be occupied by the gel during electrophoresis. The 20 space is bordered by confronting faces of the plates 14, 16 and the inner edges of the spacer strips 18, The cross-sectional view in Figure 2 depicts additional details of the plates 14, 16.
Plates 14 and 16 have inner faces, juxtaposed to one another, and outer faces which are directed away from one another. The outer and inner faces of plate 14, denoted respectively as 14a and 14b, and the outer face of plate 16, denoted as 16a, are all substantially planar and parallel to one another. The inner face of plate 16 has an elongate, shaped or recessed region, indicated in Figure 1 by hatching, extending across its upper, marginal edge, except at both lateral sides. Further in this regard, the inner face of plate 16 includes a major planar surface 16b and a step portion; the latter comprised of a minor planar surface 16c and a sloped region 16d that bridges the minor planar surface 16c and the major planar surface 16b. The major planar surface 16b and the minor planar surface 16c are substantially parallel to one another and to surfaces 14a, 14b and 16a. As also seen in Figure 2, separation medium 23 extends from the separation zone at least partially into WO 99/54721 PCTIUS99/0835 I the loading zone. above the junction between sloped region 16d and the beginning of minor planar surface 16c.
The geometry of sloped region 16d is not particularly critical, although it is preferred that region 16d be free of significant discontinuities in its surface. Thus, the sloped region 16d may meet the major and minor planar surfaces 16b and 16c at an angle such that region 16d is substantially flat), or alternatively, region 16d joins surfaces 16b and 16c by way of a smooth curve at each end of 16d. In the specific embodiment of Figure 2, the junction between the sloped region 16d and the major planar surface 16b is angled, and the junction between the sloped region 16d and the minor planar surface 16c is smoothly curved. Preferably, the curved junction between surface 16c and 16b is formed as a circular arc spanning 1 to 4 degrees, and preferably about 3 degrees. In another preferred embodiment, the length of sloped region 16d in the direction leading from the top to the bottom of the plates is preferably at least twice the depth of the recess in plate 16 the distance between surfaces 16a and 16b as measured in the direction perpendicular 15 to those surfaces). Additionally, each inner and outer face of plates 14, 16 may be beveled about the periphery so as not to present any sharp points or edges.
In an exemplary embodiment, the plate-to-plate distance between surfaces 14b and 16b is 0.2 millimeters and the plate-to-plate distance between surfaces 14b and 16c is 0.4 millimeters. However, other distances between surface 14b and surfaces 16b and 16c are also contemplated by the invention. In any case, the plate-to-plate distance separating surface 14b and surface 16b should be less than the plate-to-plate distance separating surface 14b and surface 16c. By this construction, a comb 24 0.4 millimeters face-toface) that is thick relative to the thickness of most of the electrophoresis medium layer, can be accommodated in the gap between surface 14b and surface 16c. This is because most of the electrophoresis medium layer 23 resides in the much narrower gap 0.2 millimeters) between surface 14b and surface 16b. Thus, most of the electrophoresis medium layer 23 is ultra-thin, while the comb 24 and the sample loading regions formed with the comb are comparatively thick.
As best seen in Figure 1, the top edge of plate 16 is cut away across the width of the plate, except at both lateral ends. The resulting cutout provides access for a buffer solution to make contact with the top edge of the electrophoresis medium layer.
At this point it should be noted that the region between the plate surfaces 14b and 16b is referred to herein as the separation zone, and the region between the inner surfaces of the plates in the recessed region between surface 14b and surface 16c) is referred to WO 99/54721 PCT/US99/08351 herein as the loading zone or reservoir. The region between the plate, surfaces 14b and 16d, communicating the separation zone and the loading zone; is referred to herein as the tapered portion. Additionally, the distance between the confronting surfaces of the plates 14, 16 is referred to herein as the plate-to-plate distance. As previously discussed, the plate-to-plate distance in the separation zone should be less than the plate-to-plate distance in the loading zone.
The plates 14, 16 may be made using any suitable material. In a preferred embodiment, the plates are made of a flat, chemical resistant, borosilicate glass with a low thermal expansion, such as that sold under the trade name BOROFLOAT® or TEMPAX®. In this embodiment, both sides of each plate (14, 16) should be ground and polished to remove tin from the glass surface and to generate a high degree of flatness. Computer numerically controlled (CNC) machining may then form the shaped region of the plate 16.
For example, a CNC rotary tool fitted with a silicon carbide grinding wheel may initiate ~grinding near one lateral edge of plate 16 and continue grinding while moving laterally 15 across the plate to a position near the other lateral edge. In this fashion, the shaped region can be ground in a single, continuous step. Thus formed, the shaped region has substantially uniform dimensions throughout.
Figure 3 is a front elevational view showing the comb 24 as it is set in the electrophoresis apparatus 12 of the invention. The teeth, as identified by the reference 20 numeral 26, should fit snugly in the gap 22 between the plates 14, 16, within the loading zone, so that the spaces between adjacent pairs of teeth are isolated from one another so that sample leakage between wells is prevented. As best seen in the side, cross-sectional view of Figure 4, the tips of the teeth are partly buried to a depth in the electrophoresis medium layer 23. For typical sequencing gels, the depth is preferably about 0.25 to 0.3 mm, to electrically isolate and form liquid-tight seals between adjacent sample spaces.
When the teeth have substantially uniform thicknesses in the direction perpendicular to the plate surfaces, it is preferred that the tips of the teeth do not extend beyond the junction between surface 16c and sloped region 16d, to prevent possible deformation of the teeth due to increased tightness in sloped region 16d.
In this way, each tooth 26 serves as a wall that separates two adjacent spaces, such as 28 in Figure 3, to be filled with a sample of interest. Such spaces 28 are referred to herein as loading regions. Preferably, there are at least 49 teeth extending off of the support member, and more preferably at least 97 teeth, to collectively provide at least 48 or 96 loading regions 28, respectively, with each preferably having substantially the shape of WO 99/54721 PCT/US99/08351 an inverted The upper portion of each loading region 28 extends above the upper edge of the shaped plate 16 to provide access for a sample loading implement (not shown) so that a sample aliquot may be deposited proximate the gel surface 23a.
The comb 24, as illustrated in the front and side elevational views of Figures 5 and 6, respectively, is optionally constructed of two components. One component is cut from a sheet of MYLAR® (DuPont), or another rigid, inert material, to include an elongate, central support 32, (ii) a straight-edge 34 (optional) extending off of one lateral edge of the support 32, and (iii) a series of evenly spaced teeth 26 extending off of the other lateral edge of the support 32. The material from which this component is formed should be of uniform thickness throughout. In an exemplary embodiment, this component is 0.4 millimeters thick. The other component is an elongate, semi-rigid, stop-bar 38 made from polycarbonate, or the like, which is permanently bonded to, and fully extends across the length of, an upper portion of the support 32. The upper and lower edges of the stop-bar 38 are flat, as well as the face abutting the support 32. The stop-bar 38 should be substantially 15 thicker than the support 32, the former preferably having a thickness of about 2 to 3 millimeters. By way of further illustration only, with reference to Figure 5 and the exemplary dimensions just noted for the first comb component and stop-bar 38, the exemplary comb has a length of about 20 cm along its longest dimension, and a height (from the top of straight-edge 34 to the distal ends of the teeth) of about 3.1 cm. A pull-tab 20 40 may be formed at each end of the stop bar 38 to facilitate pulling the comb 24 out from between the plates 14, 16.
Next, the teeth 26 of the comb 24 will be described. Certain terms used herein in describing the dimensions of the teeth have the following intended meanings: "width" is co-extensive with the plane of the comb and perpendicular to the longitudinal axis of a tooth, and extends from one lateral edge of the tooth to the other lateral edge of the tooth; "length" extends from the interface whereat the tooth meets the lateral edge of the support to the extreme tip of the tooth, in a direction co-extensive with the longitudinal axis of the tooth; and, "thickness" or "depth" is co-extensive with a plane perpendicular to the plane of the comb and bisecting a tooth along the tooth's longitudinal axis, and extends from one lateral face of the tooth to the other lateral face of the tooth.
In general, the comb used in practicing the invention can have any configuration that achieves a snug fit with the surfaces of the opposing plates and (ii) provides liquidtight, electrically isolated sample loading regions between adjacent teeth of the comb. A number of comb designs are known in the art and need not be detailed here. Preferably, WO 99/54721 WO 9954721PCTIUS99/083S I however, each tooth in. the comb contains a first portion beginning proximal to and extending away from the support member, such that the first region is optionally tapered, and (ii) a second, terminal portion that is more tapered than the first region and has a pointed end, for forming a liquid-tight seal with the top of the separation medium. In a more specific embodiment, each tooth has a first tapered portion which tapers gradually beginning proximate to the support member and extending along most of the tooth's longitudinal axis, and (ii) a second tapered portion, more sharply tapered than the first tapered portion, which optionally begins immediately beyond the first tapered portion and extends along the tooth's longitudinal axis to an end of the tooth distal from the support member. Such features will now be discussed with reference to the particular configuration shown in Figure 7.
As seen in the enlarged view provided in Figure 7, each tooth 26 has a gradually tapered portion 26a along most of the tooth's longitudinal axis. Here, the width of tooth 26 gradually decreases, and is thus tapered, in the direction away from the support 32.
Preferably, respective lines defined by each edge of this first tapered portion, projected out in the direction away from the support member, converge at an angle o f from 0 to degrees. Each tooth 26 also has a second tapered portion 26b that is more sharply tapered than the first portion, along its longitudinal axis beyond the shallow taper. Here, the width of tooth 26 in portion 26b decreases in the same direction, but much more rapidly than in the tapered portion 26a. As shown in Figure 7, the second, terminal tapered portion preferably ends with a terminal point at the vertex of angle .Preferably, is from about 80- to about 100 degrees. Alternatively, instead of ending at a point, the terminal portion of each tooth 26 may be blunt or slightly rounded.
One advantage of having a relatively sharp tip at the end of each tooth, such as region 26b in Figure 7, is that such sharp ends help form a liquid-tight, electrically isolated seal with the separation medium by penetrating slightly into the surface of the medium. In experiments conducted in support of the invention, the inventors have found that a good seal is significantly more difficult to achieve with blunt-ended teeth because they cannot adequately "grip" the surface of the medium so as to prevent sample cross-over between adjacent loading regions. Sharper ends, on the other hand where 8 0 degrees).
are more susceptible to bending and can lead to tearing of the separation medium.
In the particular configuration shown in Figure 7, the terminal tapered portions 26b begin immediately beyond the first portion 26a. However, the invention also contemplates embodiments in which regions 26a and 26b are separated by a third portion (not shown)' WO 99/54721 PCT/US99/0835 I having edges that are parallel or tapered. depending on design choice. Similarly, the edges in proximate portion 26a can be chosen to be parallel to each other, rather than tapered.
Preferably, however, the edges of the teeth are tapered along their entire length in the longitudinal direction, at least to some degree, in order to impart greater rigidity to the S teeth along their entire lengths, to prevent bending when the comb is inserted into the sample loading zone of the plates.
In a preferred embodiment, the teeth 26 are all of uniform thickness (in the direction perpendicular to the plate surfaces) and length. In an exemplary embodiment, the thickness of each tooth is about 0.4 millimeters or slightly greater (to ensure a snug fit with the plates), and the length of each tooth is about 10 millimeters. Further in this embodiment, the first, proximate tapered portion extends along the tooth's longitudinal axis S.a distance of about 9.7 millimeters from the support's lateral edge, and the second, more S* "sharply tapered region 26b extends along the tooth's longitudinal axis for a distance of about 0.27 millimeters beyond the first tapered portion. Additionally, and for illustrative purposes only, the width at the transition of the shallow taper and sharp taper is about 0.54 millimeters.
As shown in Figure 5, identifying means may be printed on the surface of the comb 24 to ensure that the user can clearly recognize where to inject samples. For example, numerals and/or ruled lines may be provided to indicate sample lanes 1-96. It should be appreciated that the great number of wells provided by the comb of the invention permits the loading of more samples for a single electrophoretic run than has previously been possible, which contributes to a higher throughput analysis.
Components which are not shown in the drawings are clamps used to hold the two plates together, a support structure for holding the plates in a vertical position, and electrode buffer reservoirs which contact the top and bottom edges of the gel. Such components, and their method of use, are well known in the art.
Conveniently, the apparatus of the invention can readily be retrofitted into existing equipment. It is contemplated that the apparatus of the invention should be particularly useful as a 96-lane upgrade for an ABI 377 Prism T M DNA Sequencer (Applied Biosystems Division of The Perkin-Elmer Corporation).
Another aspect of the invention provides a method for separating bio-organic molecules using a slab gel. Preferably, the method is practiced in connection with an electrophoresis apparatus as described above. The method is particularly useful for the electrophoretic separation of nucleid acids, such as DNA, using an ultra-thin gel.
14 WO 99/54721 PCTUS99/08351 In a preferred embodiment, the method is practiced with a slab gel electrophoresis apparatus having a pair of spaced, confronting plates defining a multi-lane separation zone adapted to hold a separation medium and an upper loading zone, and a comb having a plurality of teeth adapted to be inserted into the loading zone for holding samples and S defining sample lanes. According to the method, the comb is inserted into the loading zone so that the teeth abut an upper portion of the separation medium, thereby forming a series of fluid-tight, elongate sample-loading apertures of substantially rectangular cross-section having a plate-to-plate width greater than that of the inter-plate distance in the separation zone.
With reference to the figures, the plates 14, 16 are placed face-to-face so that the shaped region of the plate 16 confronts the inner face 14b of plate 14. Spacers strips 18, are interposed at both vertical edges to establish a gap 22 between the plates 14, 16. As previously discussed, the gap 22, in turn, determines the width for the interposed gel 23.
Thus, if it is desired to form an ultra-thin gel having a thickness of 0.2 mm within the separation zone, then 0.2 mm spacer strips should be employed. Once the spacer strips 18, are in place, the plates 14, 16 are then compressed together by any suitable means (not shown), which could include any of various type of clamps or clips along their lateral edges.
A seal (not shown) is placed along the open top edge of the confronting plates 14, 9...I 16. In a preferred embodiment, the seal is provided by straight edge 34 of comb 24 (Fig. which is dimensioned to leave small apertures on either side of the comb, in order for air to escape during filling of the gel. In this regard, comb 24 is inverted (with the teeth 26 :pointing away from the plates) and its straight edge 34 is pushed down between the plates 14, 16 before the gel solution is poured into the gap 22. The stop bar 38, being substantially wider than the gap 22 along the lower open edge of the plates 14, 16, ensures that the straight edge 34 extends down to the desired depth uniformly from one lateral side of the loading zone to the other lateral side. The straight edge 34 thus serves to establish both the straightness and height of the upper edge 23a of the gel 23.
A separation medium, such as a polyacrylamide gel solution, is prepared and poured into the lower open end of the plates, until the entire separation zone is filled. The medium is then allowed to solidify.
After the gel 23 has solidified, comb 24 is removed and inverted to the position shown in Figures 2 and 3 of the drawings. The tips of the teeth 26 should be pressed down into the gel so that they become partly buried to a depth in the gel 23. Preferably, the WO 99/54721 PCT/US99/0835 I depth is about 0.25 0.3 mm. In this way, each tooth 26 serves as a wall that separates two adjacent spaces, or loading regions, such as 28, to be filled with a sample of interest.
The upper portions of the loading regions 28 between the teeth 26 should extend above the upper edge of the shaped plate 16 to provide access for a sample loading implement so that sample aliquots may be deposited proximate the gel surface 23a. In Fig. 3, for example, about 70% of the length of the teeth extend into the loading zone of the plates, and the other 30% remains outside of the plates. This ensures adequate sample volumes on the order of 1.5 microliters) in the loading zone while at the same time providing access for the tip of a micropipette to load the sample loading regions (sample wells). The tip of the micropipette is carefully lowered through the U-shaped region between adjacent teeth to just above the separation medium, and the sample is gradually released into the sample well while the micropipette is simultaneously withdrawn.
o Next, the top and bottom of the polymerized gel are placed in electrical contact ooeo with respective buffer reservoirs (not shown). For the upper buffer reservoir, the inner, upper region of plate 14 and the comb 24 form one side wall, and the U-shaped spaces extending above the upper edge of the shaped plate 16 will be entirely immersed in the upper buffer solution.
Samples are loaded into the loading regions 28 along the upper edge of the gel 23.
In this regard, the recessed region of plate 16 facilitates the depositing of samples into the loading regions 28 by permitting insertion of a sample-loader tip (not shown) having an outer diameter substantially greater than the inter-plate distance within the separation zone.
In a preferred embodiment, the inter-plate width within the shaped region allows a loader tip of nearly 0.4 mm to be inserted between the plates 14, 16 for depositing samples proximate the gel surface. Conveniently, conventional pipettes can be used with the apparatus of the invention to load samples onto ultra-thin gels.
Subsequent to loading samples, an electric field is applied across the gel 23 and the samples are allowed sufficient time to migrate down the gel 23. As electrophoresis proceeds, the edge-to-edge width of each tooth 26, as measured at the gel surface 23a, establishes the dark space that will separate adjacent lanes corresponding to each sample.
In this regard, since the present invention provides a greater tooth width at this interface than previous shark's tooth combs, the problem of insufficient dark space between adjacent lanes, often encountered with combs having very narrow teeth, is avoided.
At the end of the electrophoretic run, the plates 14, 16 are pried apart to expose the gel. The gel can then be dried and the results visualized, for example, by autoradiography.
16 WO 99/54721 PCT/US99/0835 1 In light of the description above and the accompanying drawings, it can be seen how the various objects of the invention are met. By providing an elongate recess in the sample loading zone of at least one of the two opposing electrophoresis plates, the present invention provides a way to increase the loadable volume of the samples while S simultaneously facilitating the otherwise difficult loading of samples using conventional ultra-thin pipettes. Further, by providing a loading zone with parallel opposing plate surfaces having a thickness greater than the separation medium, the plates of the invention promote a snug fit between the teeth of the comb and the plates, so that sample cross-over is effectively suppressed. The wider sample loading region also enables lanes to be placed more closely together since narrowing of the lateral spacing between wells is offset by greater thickness of the wells in the direction perpendicular to the surfaces of the glass plates, and (ii) the increased thickness dimension lends increased rigidity to the teeth of the oooo comb, so that narrower teeth can be used. Finally, by providing a comb embodiment wherein the spacing between edges of adjacent teeth at their distal end regions where the *0.
teeth cross the surface of the separation medium is a multiple less than 3 of the width of the individual teeth where the teeth cross the surface of the separation medium, the invention provides cleanly separated lanes with sufficient separation between lanes so that coalescence of sample bands from adjacent lanes is significantly avoided. Furthermore, the S:i" present invention dramatically increases the lane density that can be achieved in electrophoretic applications such as nucleic acid sequencing, by providing combs that afford lane densities of greater than about 5 lanes per centimeter, roughly doubling o:oo previous standard lane densities.
The present invention further provides procedures useful for optimizing resolution, particularly in connection with those embodiments providing a relatively large number of sample loading regions greater than 64) on a single gel. Such optimization procedures include: reducing the amount of nucleic acid loaded in each lane as compared to the amount loaded for instruments having fewer loading regions 64 or fewer); minimizing the concentration of salts in each sample; loading each sample very close to the bottom of the sample loading region (sample well); and/or utilizing a unique loading protocol, taught herein, in which the sample wells contain water, or a low ionic strength buffer 0 0.5X TBE, or other buffer of equivalent ionic strength).
when the nucleic acid samples are loaded. These procedures, aspects of which can be found in the ABI PRISM 377 DNA Sequencer With 96-Lane Upgrade User Bulletin [PE WO 99/54721 PCT/US99/0835
I
Applied Biosystems (Foster City., incorporated herein by reference, are discussed below.
In some cases, resolution can be increased by using a decreased amount of nucleic acid per sample well. For example, to lower the amount of DNA in each lane, a user can use less DNA in sequencing reactions and increase the number of cycles; resuspend the sample in a larger volume of loading buffer; and/or load between 0.5-1 PL of the sample in each well. A user can optimize his or her resuspension and loading volumes by doing a series of titrations, and running gels to determine the best combination of signal strength and resolution.
On some occasions, excess salts in a sample can lead to lowered resolution. Salts can accumulate in a sample, for example, when the loading buffer of the\cycle sequencing reactions has a high concentration of salts, and/or when salts are used to purify extension products from a cycle sequencing reaction. Thus, in certain situations, the present invention contemplates a reduction (minimization) of the concentration of salts in a sample.
In some cases, resolution can be improved by loading samples close to the gel surface rather than from the top of the well. One embodiment for loading samples, for example, contemplates that a user: expel urea leached into the wells by rinsing the wells sufficiently with either: ddH20 (if using the loading protocol described below); or IX TBE 20 if using a standard protocol; draw sample(s) into a tip or multi-channel loader with a needle diameter of 0.2-0.25 mm. (Suitable multi-channel loaders include those made by Kloehn (Las Vegas, NV) and World Precision Instruments (Sarasota, position the tip(s) close to the bottom of the well (gel surface); and, very slowly dispense up to 1 PL of sample into each well, withdrawing the syringe tip as the sample is being expelled.
Particularly for 96 lane gels, one embodiment contemplates the loading of no more than about 0.5 UL of sample per lane.
As previously indicated, the present invention provides a loading protocol that can be used to increase resolution. In certain situations, the loading protocol of the invention can be useful to minimize band broadening and to improve stacking. Preferably, according to the loading protocol, the upper buffer reservoir contains deionized water when the nucleic acid samples, DNA, are loaded into the sample wells. If the use of deionized water fails to provide adequate results, then a buffer solution of low ionic strength can be used instead. In one embodiment, the ionic strength of the buffer solution falls within a range equivalent to that of 0 0.5X TBE, and more preferably 0 0.2X TBE. After the 18 WO 99/54721 PCT/US99/08351 samples have been loaded, the final concentration of buffer in the buffer reservoirs is increased, made up to that of about IX TBE.
As is well known, ionic strength is a charge-weighted measure of the total concentration of all ions in a solution. For an electrolyte solution, ionic strength is given by: I=J 2 where Z is the charge on ionic species i and C is the molal concentration, the summation being continued over all the different ionic species in the solution (See, for example, J.
Butler, Ionic Equilibrium, A Mathematical Approach, Addison-Wesley Publishing 10 Company (Reading, Massachusetts), 1964). Using well-defined principles, such as this, those skilled in the art can readily choose buffers and concentrations therefor exhibiting an ionic strength like that of the TBE solutions of the preferred concentrations specified herein. Exemplary buffers include physiological buffers and low salt buffers typically used with DNA TE, TAE, TBE, TTE, phosphate buffers, etc.). Instead of, or in addition 15 to, using ionic strength to define suitable buffers, particular buffers and concentrations can be chosen to have a salt concentration and/or conductivity like that of the TBE solutions of the preferred concentrations specified herein.
One embodiment of the loading protocol, particularly useful for optimizing the resolution of DNA separations, includes the following steps: 1) A separation medium, such as a polyacrylamide gel solution, is prepared, then poured and allowed to polymerize for about two hours). These initial steps can be carried out substantially in accordance with standard methods, such as described above (Also see, for example, Sambrook, Fritsch, E. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989). One preferred gel solution, contemplated for use herein is a 5.25% PAGE- PLUS gel (Amresco Inc. (Solon, Other exemplary gel solutions, contemplated for use herein, are set out in the ABI PRISM 377 DNA Sequencer User's Manual, Appendix A-Gel Recipes Quick Reference Guide [PE Applied Biosystems (Foster City, also see the User Bulletin ABI PRISM 377 DNA Sequencer, Improved Gel Formulations for Extended Sequencing Read Lengths [PE Applied Biosystems (Foster City, both incorporated herein by reference. A straight edge can be employed to establish the straighmess and height of the edge of the gel.
WO 99/54721 PCT/US99/0835 2) Upon removing the straight edge, the sample loading region is cleaned of gel fragments. Care is taken to ensure that substantially all gel fragments are removed from the loading region prior to inserting the teeth of a comb.
3) A comb (such as taught above) is then inserted, teeth first, into the sample loading region. Preferably, no liquids other than water are in the comb region when inserting the comb. Unlike many standard protocols, IX TBE should not be added at this point to ease the comb in.
4) Next, the gel is mounted in an appropriate support structure a vertical cassette).
5) At this point, a plate check can be performed to ensure that the plates and gel are clean and free of contaminants fluorescent species) before the samples are loaded. Exemplary instructions for performing a plate check are set forth in the ABI PRISM 377 DNA Sequencer User's Manual, Chapter 3-Instrument Operation [PE Applied Biosystems (Foster City, incorporated herein by reference.
15 6) Deionized water is then added to the upper buffer reservoir, preferably up to about 90% of the total reservoir volume. For example, for a buffer reservoir having a total volume of 600 mL, about 540 mL of dHO can be added.
Or, for a buffer reservoir having a total volume of 660 mL, about 594 mL of 20 can be added. It should be noted that results may vary depending on local variations in water quality. In the absence of a sufficient stagger between odd and even lanes, the same amount of 0.25-0.5X TBE solution can be substituted in place of the deionized water in the upper buffer reservoir. This solution must be made up to IX TBE in step 14 of this procedure.
The lower buffer reservoir is filled with 1X TBE with the ABI PRISM 377 DNA Sequencer [PE Applied Biosystems (Foster City, CA)], approximately 600 mL are added up to the over flow line).
7) A prerun is next started, and then immediately paused. Once started and paused, the prerun continues to bring the gel up to run temperature about 51 No electrophoresis takes place. Exemplary instructions for a prerun are set forth in the ABI PRISM 377 DNA Sequencer User's Manual, Chapter 3-Instrument Operation [PE Applied Biosystems (Foster City, incorporated herein by reference.
8) Next, the loading region is rinsed well. This can be done using a syringe of water with a needle on the end of it, and dragging the needle along the ledge in WO 99/54721 PCT/US99/08351 front of the comb while dispensing the water. During this operation. it is important to avoid touching the comb with the needle.
9) The odd lanes are then loaded. Preferably, this is accomplished by positioning the tip(s) close to the bottom of the well (proximate to the gel surface), and very slowly dispensing up to 1 pL of sample into each well, withdrawing the syringe tip as the sample is being expelled.
Another prerun is started, then paused after 2 minutes.
11) The loading region is then rinsed again, as above (step 8).
12) Next, the even lanes are loaded, using the technique described above in step 9.
13) The prerun is resumed for 1.5 minutes, and then canceled.
14) 10X TBE is then carefully added to the upper buffer reservoir to get a final volume of IX TBE. For example, if 540 mL of water was used in step 6, above, then add 60 mL of 10X TBE to get 600 mL of lX TBE. Or, if 594 mL of water was used in step 6, 15 above, then add 66 mL of 1OX TBE to get 660 mL of 1X TBE.
Next, the contents of the upper buffer reservoir are mixed, using a clean 50-mL syringe.
16) The run can then be started.
The just-described embodiment of the loading protocol of the invention is 20 especially useful for optimizing resolution in connection with gels having a high lane density, 96 lanes or more. In one preferred embodiment, plates that provide an expanded loading region, as taught herein, are employed. In addition to increasing resolution, the above protocol can be useful for achieving a longer read length than that obtainable using standard methods. Especially good results, in these regards, can be realized using the above procedure in connection with an ABI PRISM® 377 DNA Sequencer (with the 96-lane upgrade) employing 48-cm well-to-read (WTR) plates.
In another embodiment, similar to the just-described embodiment, TTE buffer is used instead of TBE. In brief, samples are loaded into sample wells that contain water or a low ionic strength TTE buffer (in a fashion similar to the above embodiment). After the samples have been loaded, the ionic strength in the upper reservoir is increased to that of 1OX TTE buffer. Then separation by electrophoresis is carried out. Exemplary steps, described in connection with the ABI PRISM® 377 DNA Sequencer (with the 96-lane upgrade) [PE Applied Biosystems (Foster City, are as follows: WO 99/54721 PCT/US99/0835 1) One liter of 10X TTE buffer is made by combining the following: Tris-base, 60.55 grams; (ii) 0.5M TAPS, 121.65 grams (Sigma part number T5130 [Sigma: St. Louis, Missouri, USA]), (iii) 10mM EDTA, 3.72 grams, and (iv) deionized water to IL.
mixed well. Preferably, the pH of the buffer is about 8.3 0.2.
2) A 4.25% PAGE-PLUS gel is prepared by combining the following components: 40% PAGE-PLUS, 5.3 ml; (ii) urea, 18 grams, (iii) 6 ml 10X TTE [note, this is 1 ml more than the standard protocol, 1.2 X buffer in the gel rather than IX in a standard gel]; (iv) deionized water to 50 ml; mixed and polymerized with TEMED, p.l, and (vi) 10% APS (freshly prepared), 250 tl. Preferably, the gel is prepared employing 48-cm well-to-read (WTR) plates.
3) The gel, so prepared, is then loaded onto the instrument with IX TTE in the lower buffer reservoir and water (preferably deionized water) in the upper buffer reservoir.
It is important to accurately measure the amount of water added to the upper buffer chamber, since, in a later step, a more concentrated TTE solution 10 X) will be added 15 to bring the final TTE concentration up to about 2X. In a typical operation, for example, 520 ml of water is added to an upper buffer chamber having a capacity of about 650 ml.
oo Next, a prerun is started, and then immediately paused to bring the gel up to the correct temperature. Note, a complete prerun is not performed.
A 48-cm run module is then selected, suitable for the chemistry at hand, and 20 the following changes made: Temperature: 45° C Power: 40W limiting Voltage: 4000V Amps: RunTime: 14-18 hours (It should be noted that the actual run conditions should be 40W constant power, and approximately 1800V and 22mA. The power will be kept constant at 40W due to the influences of the other electrophoresis parameters).
6) A sample sheet is then prepared by setting up the sample sheet and analysis defaults. Base-calling software is available from PE Applied Biosystems (Foster City, CA). Preferred software is distributed as "377 Long Read (LR) Base Caller." 7) The wells are then rinsed.
8) Next, the samples are loaded into the odd lanes.
9) The prerun is then resumed for 2 minutes, then paused.
WO 99/54721 PCT/US99/0835 Next. the even lanes are loaded and the prerun is resumed for 1.5 minutes.
then canceled.
11) 10X TTE is added to the upper buffer reservoir in an amount sufficient to bring the concentration up to 2X TTE. For example, if 520 ml of water was added in step 3 (above), then 130 ml of OX TTE is added at this point. The solution is then mixed gently with a clean 50 ml syringe. Care should be taken not to spill any of the buffer, as the correct buffer concentration can be very important to the success of this gel formulation.
12) The altered run module, set up in step 5 (above), is then started.
The just-described embodiment of the loading protocol of the invention is especially useful for optimizing resolution and obtaining long read lengths in connection with gels having a high lane density, 96 lanes or more. In one preferred embodiment, S plates that provide an expanded loading region, as taught herein, are employed. Tests carried out in support of the present invention, using the just-described protocol, have 15 produced >1000 base reads.
In addition to the above procedures, the present invention also provides a method useful for achieving long read lengths in connection with embodiments providing relatively fewer sample loading regions 64 or fewer) on a single gel.
Another aspect of the present invention provides a procedure for obtaining longer 20 base reads than have been obtainable in the past. Generally, according to one embodiment, a 48 cm well to read (WTR), 36 lane 4.25% PAGE-PLUS gels and TTE buffer. This embodiment is especially useful in connection with gels having 64 lares, or fewer 36, 48 and 64 lane gels).
One particular protocol, which calls for 36 lanes with TTE buffer and 4.25% Page- Plus gels, includes the following steps: 1) One liter of 1OX TTE buffer is made by combining the following: Tris-base, 60.55 grams; (ii) 0.5M TAPS, 121.65 grams (Sigma part number T5130), (iii) EDTA, 3.72 grams, and (iv) deionized water to 1L; mixed well. Preferably, the pH of the buffer is about 8.3 0.2.
2) A 4.25% PAGE-PLUS gel is prepared by combining the following components: 40% PAGE-PLUS, 5.3 ml; (ii) urea. 18 grams, 6 ml 10X TTE; (iii) deionized water to 50 ml; mixed and polymerized with (iv) TEMED, 25 pl, and APS (freshly prepared), 250 pl. Preferably, the gel is prepared employing 48-cm well-toread (WTR) plates.
WO 99/54721 PCT/US99/08351 3) The gel is assembled with 2X TTE in the upper buffer chamber and IX TTE in the lower buffer chamber (a heat plate is not necessary).
4) A 48-cm run module is then selected, suitable for the chemistry at hand, and the following changes made: Temperature: 450 C Power: 40W limiting Voltage: 4000V Amps: Run Time: 14-18 hours (It should be noted that the actual run conditions should be 40W constant power, and approximately 1800V and 22mA. The power will be kept constant at 40W due to the influences of the other electrophoresis parameters.) 5) A sample sheet is then prepared by setting up the sample sheet and analysis defaults. Current basecallers that allow 32000 scans to be analyzed part of the 15 Sequencing Analysis 3.2 and 3.3 releases) are preferably employed. Base-calling software is available from PE Applied Biosystems (Foster City, CA). While the ABI50 basecaller generally works quite well with this data, the semiadaptive basecaller occasionally gives better results. The data should be analyzed with both to determine which gives the best data.
S 20 6) The samples are loaded, and the altered run module, set up in step 4 (above), started. Preferably, when loading samples, the loading volume for the DNA is kept relatively small. For gels with less than 96 lanes, one exemplary embodiment contemplates the use of no more than about 1 pl.
This just-described protocol works particularly well with 36 lane gels, though it can be used with other configurations, as well (such as 48 and 64 lane gels). The inter-plate distance in the sample wells (loading regions), in the just-described embodiment, is preferably substantially the same as that within the separation zone the loading region is not expanded). For example, each of the confronting plate surfaces can be substantially planar, and these confronting planar surfaces can be substantially parallel to one another as found in many standard slab gel arrangements. A 14 hour run generally provides 1000 bases of data. To collect more data, the gel can be run longer.
Those skilled in the art can now appreciate from the foregoing description that the broad teachings of the present invention can be implemented in a variety of forms.
WO 99/54721 PCT/US99/08351 Therefore, while this invention has been described in connection with particular embodiments and examples thereof, the true scope of the invention should not be so limited. Various changes and modification may be made without departing from the scope of the invention, as defined by the appended claims.
*O O*O *oo

Claims (8)

1. A slab gel electrophoresis apparatus having a pair of spaced, confronting plates defining a multi-lane separation zone adapted to hold a separation medium and an elongate upper loading zone, and a separation medium held within said separation zone; further comprising: a comb having an elongate support member and a series of teeth extending from one edge of said support member; wherein each tooth has a first tapered portion which tapers gradually io beginning proximate to said support member and extending along at least half of the tooth's longitudinal axis, and (ii) a second tapered portion, more sharply tapered than said first tapered portion, which begins immediately beyond said first tapered portion and extends along the tooth's longitudinal axis to an end of the tooth distal from said support member; wherein said teeth are positioned between said plates in said loading zone with the distal end of each tooth penetrating said separation medium, thereby forming a plurality of elongate loading regions, between adjacent teeth, that communicate with the separation zone. 20
2. An apparatus according to claim 1, whereby said teeth have front and rear surfaces that are substantially planar and parallel to one another.
3. An apparatus according to claim 2, wherein said separation medium within said separation zone is less than 0.3 mm thick, and wherein said teeth of said comb are greater than 0.3 mm thick.
4. An apparatus according to claim 3, wherein the face-to-face thickness of S: the comb is 1.5 to 3 times greater than the plate-to-plate distance within said separation zone.
An apparatus according to claim 2, wherein at least 97 teeth extend off said Z! 4dge of said support. ':\fionvNKJ\Spcmis\div o"35656.doc -27-
6. An apparatus according to claim 2, wherein each loading region has substantially the shape of an inverted
7. An apparatus according to claim 1, wherein at least one of said plates has an elongate recess formed in the upper portion thereof, said recess forming, together with the confronting plate, an elongate upper loading zone having a plate to plate distance which is substantially constant throughout said upper loading zone and (ii) is greater than the plate-to-plate distance within the separation zone; said plate further comprising an elongate tapered portion joining the separation zone with the loading zone, said elongate tapered portion having a plate-to-plate distance that progressively decreases from the loading zone to the separation zone; and wherein said separation medium extends at least partially into said tapered portion.
8. A slab gel electrophoresis apparatus having a .pair of spaced, confronting plates defining a multi-lane separation zone adapted to hold a separation medium and an elongate upper loading zone, and a separation medium held within said separation zone; a comb comprising: ooe• :an elongate support member; a series of teeth extending from one edge of said support member; g wherein said teeth are positioned between said confronting plates in said loading zone with their distal end regions penetrating a surface of said separation medium; .Iwherein said teeth and confronting plates define a plurality of loading .o 25 regions, between adjacent teeth, that communicate with the separation zone; 0wherein said teeth have front and rear surfaces that are substantially planar and parallel to one another; wherein the face-to-face thickness of the comb is a multiple greater than 1.1 of the plate-to-plate distance in the separation zone. DATED: 3 April, 2002 CORPORATION (NY)
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US5073246A (en) * 1990-05-16 1991-12-17 Bio-Rad Laboratories, Inc. Slab electrophoresis system with improved sample wells and cooling mechanism
WO1997043630A1 (en) * 1996-05-17 1997-11-20 Purdue Research Foundation Miniaturized disposable gels for dna analysis

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0285394A2 (en) * 1987-03-30 1988-10-05 Fuji Photo Film Co., Ltd. Electrophoresis device
US5073246A (en) * 1990-05-16 1991-12-17 Bio-Rad Laboratories, Inc. Slab electrophoresis system with improved sample wells and cooling mechanism
WO1997043630A1 (en) * 1996-05-17 1997-11-20 Purdue Research Foundation Miniaturized disposable gels for dna analysis

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