CA2187097A1 - Control of temperature gradients during gel electrophoresis using turbulent coolant gas flow - Google Patents
Control of temperature gradients during gel electrophoresis using turbulent coolant gas flowInfo
- Publication number
- CA2187097A1 CA2187097A1 CA 2187097 CA2187097A CA2187097A1 CA 2187097 A1 CA2187097 A1 CA 2187097A1 CA 2187097 CA2187097 CA 2187097 CA 2187097 A CA2187097 A CA 2187097A CA 2187097 A1 CA2187097 A1 CA 2187097A1
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- Prior art keywords
- exchange medium
- gel
- gaseous heat
- impingement
- plate
- Prior art date
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44708—Cooling
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- Life Sciences & Earth Sciences (AREA)
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- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
A gel electrophoresis separation apparatus comprising, in combination, a gaseous heat-exchange medium, a gaseous heat-exchange medium driving means, and an impingement means, whereby the gaseous heat-exhange medium is driven by the gaseous heat-exchange medium driving means across the impingement means to provide a flow of the gaseous heat-exchange medium on the surface of the gel plates, whereby the flow induced by passage of the gaseous heat-exchange medium through the impingement means thereby minimizes temperature gradients within the gel by forced convection.
Description
Wo 95127198 7~97 CONTROL OF TEMPERATURE GRADIENTS DURING GEL
ELECTROPHORESIS USING TURBULENT COOLANT GAS FLOW
BACKGROUND OF THE INVENTION
1. Field of the Invention This inv~ntion relates to the separation of single-stranded DNA fragments by slab gel el~ opl.~..,,is in performing DNA sequencing. Specifically, the invention relates to an apparatus for performing DNA sequence analysis by slab gcl elc.,LI~,vh~.c~iswhereintheapparatuscomprisesaturbulent-flow,gasheat-exchange medium impingement-based temperature controlling subassembly. A particular feature of the invention is that thermal temperature gradients generated in the gel separation media during el~ ,o~,l.o~ is are -' The speed, reliability and readability of DNA sequence analysis using the apparatus of the invention is thereby improved.
ELECTROPHORESIS USING TURBULENT COOLANT GAS FLOW
BACKGROUND OF THE INVENTION
1. Field of the Invention This inv~ntion relates to the separation of single-stranded DNA fragments by slab gel el~ opl.~..,,is in performing DNA sequencing. Specifically, the invention relates to an apparatus for performing DNA sequence analysis by slab gcl elc.,LI~,vh~.c~iswhereintheapparatuscomprisesaturbulent-flow,gasheat-exchange medium impingement-based temperature controlling subassembly. A particular feature of the invention is that thermal temperature gradients generated in the gel separation media during el~ ,o~,l.o~ is are -' The speed, reliability and readability of DNA sequence analysis using the apparatus of the invention is thereby improved.
2. Descrirtion of the Prior Art Gel electrophoresis is a fundamental biochemical separation techniquc thaî
forms the basis for distinguishing a variety of biologically important molccules on the basis of size, charge or a combination thereof. Specific examples of biological molecules advantageously separated by gel electrophoresis include proteins and nucleic aids. Electrophoresis is usually performed in a gellcd (e.g., agarose) or polymeri~ed (e.g., polyacrylamide) media (generically termed a "gel") that contains an electrically conducting buffer. Electrophoresis is performed wherein a voltage ~5 is applied ~ia chemically inert metal electrodes across the cross-sectional arca of the gel. The biological sample of interest is placed into pre-formed sample wells in the I
WO 95/27198 r~.J o l~ -2 1 ~7097 8el. usually at one end of the gel, and the polarity of the applied voltage is arrangcd so that the biological sample migrates through the gel towards one of the electrodes (usually positioned at the opposite end of the gel from the samples). Wherc appropriate, the inverse linear r~ ' ir between migration distance and molecular size is maintained by the addition of chemical denaturants (such as urea, formamide, or sodium dodecyl sulfate) to the gel and cle~,LIu,uh~le~i~ buffer.
A particular application of gel electrophoresis is the separation Or single-stranded DNA fragments in the determination of the nucleotide sequence of a nucleic acid of interest. To this end, a collection of single-stranded DNA fragments is generated either by chemical degradation of the nucleic acid (using the Gilbert method, see e~e" Maxam and Gilbert (1980), Methods Enzyme, ~, p499-5ûO) or by replacement DNA synthesis using a polymerase (using the Sanger method, see e.e.,Sanger, F., Niklen, S., and Coulson, A.R. (1977) Proc. Nat. Acad. Sci. USA 74~ p5463-5467). This co11ection of single stranded DNA fragments includes a fra8ment corresponding to each position in the sequence to be determined; in the most frequently-used sequencing method, this correspondence is directly related to thc distance from a fixed site of initiation of polymerization at a primer that is annealed to the nucleic acid to be sequenced. Thus, determination of the desiredsequence depends on the separation of each of the fragments, which differ in length by only a single nucleotide.
Traditionally, the identity of each of the possible nucleotides at eacb position(adenine, guanine, cytosine or thymidine) is distinguishcd by performing a sequencing reaction specific for each endin8 nucleotide in separate chemical reaction mixtures. Thus, each sequencing experiment is typically perrormcd in 4 separate tubes, wherein are generated a collection of fragmen~s each ending at a W0 95127198 1'~ r ~ --t ~ 2 1 ~370q7 position corresponding to the terminating nucleotide used in that reaction. A
nucleotide sequence is thereafter determined by performing denaturin8 gcl ele~.LIcrho.~is on each of a set of 4 reactions, each reaction electrophoresed individually in adjacent lanes of a single sequencing gel. The presence of a band S at a position in a nucleotide-specific lane of such a gel indicates the identity of that nucleotide at that position in the sequence. Using conventional techniques, each of the fragments is r~tiic~i~b~ , and the bands are visualized after electrophoresis by autoradiography.
Alternatively, recent t~rhnnlogi~ plu~c~cnts have resulted in the development of automated sequencing machines. Such machines contain a spectrophotometric detection means at the distal end of the gel opposite to the loading wells. The DNA fragments are synthesized using either a fluorcscent-labeled primer or fluorescent-labeled nucleotides. In either case, each of thc nucleotide reactions continue to be performed separately, wherein the fragments corresponding to each of the nucleotides is fluorescently labeled with a distinguishable fluorescent label. However, because each fluorescent label exhibits fluorescent emission at a characteristic frequency, it is possible to distinguish thc identity of the nucleotide at the terminal position of each fragment from its fluorescence emission signature. This feature allows the automated collection ornucleotide sequence information spectrophotometrically using these devices. In addition, the existence of distinguishable fluorescent signatures for cach of thc fragments allows the products of the four separate sequencing renctions to be electrophoresed in a single lane of the gel.
These developments have increased the requirements for accuratc and reliable separation of the sequencing fragments generated in nucleotide scquencing WO 95/27198 ,: ~C ~ ' reactions. However, there are a number of technical limitations inherenl in current slab gel elc~,~.uv}.o.cs; technology that have remain unsolved in the current state of this art. For example, limitations on the range of distinguishably-separable fragments necessitates that each sequencing reaction series be loaded onto the gel in two sets of lanes loaded at two separate times. The lanes loaded first (and thus elc~ uvho~c~cdlongest)areusedtodeterminethenucleotidesequencefurthestfrom the polymerization initiation site (and are thus the ~&Ç5~ fragments), while thesecond-loaded set are read to determine the nucleotide sequence closest to the initiation site. The relative timing of loading and the time the reactions are aliowed to elc.,~.uvl-lo~ , are empirically chosen so that these ranges overlap; thus, one entire section of any particular nucleotide sequence can be read. The result of these limits of this CIC~ ù~ Ol~ iS that a maximum of 350-400 bases can be read from a s~ ing, reaction that is initiated at a given site. Thus, the development of methods and apparatuses capable of resolving a longer extent of nucleûtide sequencc than is currently possible would be a useful development in this art.
A number of other technical constraints that limit the extent ûf nucleotide sequence information that can be obtained using conventional electrophoresis techniques are the consequence of the electrophoretic conditions needcd for DNA
sequence analysis using slab gel electrophoresis. Preeminent among these arc thermal temperature gradients produced within the gel during electrophoresis.
In order to separate the single-stranded DNA fragments during electrophoresis proportional to the length of each fragment, a potential Or 1000-3000 volts [(1-3 kilovolts (kV)] is typically applied across the gel for 2-16 hours.
Approximately 30 to 60 watts of power is dissipated uniformly in the gel as heat and then from the surface of the gel plates zontaining the slab gel. Such heating is WO 95/2~198 r~ u~
j~, f;~ 21~9~
useful (and, in fact, nccessary), because it promotes n~oin~rnsnr~ of the single-stranded DNA in the denatured state (so that migration distancc is inverscly proportional to fragment length). However, the extent of such heating is limited to the thermal tolerance of the materials comprising the electrophoresis apparatus.S This limitation places an upper limit on the magnitude of the applied voltage; since the duration of cle~llu~ ,sis depends on the magnitude of the applied voltage, the capacity for decreasing the duration of ele~,l.u,uh~ .is is also constrained by the limitation of the magnitude of the applied power.
An additional problem associated with electrogenic heating of DNA
lû sequencing gels is the uneven distribution of such heat throughout the gel, resulting in thermal temperature gradients in the gel. Under conventional denaturing acrylamide gel ele~ opl.ol~,~is conditions, the central one-quarter to one-third of thc gel becomes warmer during electrophoresis than the rest of the gel throughout its length. Because the mobility of the single stranded DNA fragmcnts varies proportionally with temperature (at an increasing rate of approximately 2.2% perC), the col~s~u,.~,llc~ of uneven heating is that the samples in the center lanes run faster than the samples on the edges. The result is the production of sequencing gels wherein the pattern of bands in the lanes across the gel gives the appearance Or"smiling": the bands are u-o~-~,ii,iv~ily shifted anodally (i.e. upwards using conventional techniques) going from the center of the gel towards the edges.
Another serious problem to which there was no solution proposcd or implemented in the prior art is the problem associated with high-voltagc slab gel electrophoresis -- the gencration of front-to-back thcrmal tcmperature gradients. In particular, the resolution of each of thc bands from all othcr bands rcsolvcd in cach lanc of a sequencing gel is affected by thcsc typcs of thcrmal gradicnts.
WO 95/27198 ~ r P~~ O -r Band resolution depends on the size (i.e., the thickness) of each band, as weil as the relationship between the average thickness of each band and the width of thc space separating each band. A sequencing ladder comprised Of thick bands will contain fewer resoivable bands on average than a gel having thinner, more tightly-resolved bands, simply due tO the finite length of the resolvable portion of the gel.
Also, since each of the bands in a sequeneing gel ean in principle be separ~ated by as few as one nucleotide, stretches Of DNA having multiple repeats Of a particular nucleotide (e.g., 5'-11 1 1 11 1 1 1-3') will be more diffieult (if not impossible) tO
resolve in a gel haYing thicker, less tightly-resolved bands than in a gel having thinner, more tightly-resolved bands. Although this factor is particularly importanl for resolving repetitive stretches Of one type of nucleotide (because the phenomenon isexacerbatedbycl~ opl.o-cslsinthesamelaneofsingle-strandedDNAfragmcnts eorresponding to eaeh of these nucleotides), the problems of band resolution that are band width-related also occur in certain portions Of a gel across more than one gel lane (so-ealled "compression zones").
These problems have been reeognized in the prior art, as is evidenced by the use of [35S]-labeled deoxynucleotides for labeling the DNA in sequencing reactions (see Sambrook e~ al., il7id.). The bandwidths of autoradiographic bands producedfrom ~-partiele emissions from 35S are narrower than the bandwidths produced rrom sZP f-particle emissions beeause ~IsS emits lower-energy particles that convert silver grains over a narrower area in the autoradiographic film. The userulness Of suchnarrower bandwidths is evidenced by the acceptance of this method in the art in thc face of a number of practical disadvantages associated with 35S usc. These includc ~onger autoradiographic development t ~.
~ Wo 95~27198 ~) 1 8 7 ~ ~ 7 . ~ ,a~
A major determinant of the band-width of single-stranded DNA fragments separated by slab gel elecl-opk~ is using high voltages is the generation Or rront-to-back thermal temperature gradients in the gels during high-voltage electrophoresis. Thus, there was an unappreciated and unfulfilled need in the DNA
sequencing/ gel cle~L.o~,h~lc~is art for an apparatus for performing high-voltage, slab gel electrophoresis under conditions in which both front-to-back as well as side-to-side thermal temperature gradients are min' ' Thus, limitations of the prior art include: limits on how high the voltage can be, due to overheating of the gels and thereby resulting in longer run times; limits on ionic strength since higher ionic strengths need higher power; and unevcn heating, causing "smiling" (i.e., left-to-right) distortion and decreased resolution (front-to-back).
Attempts to minimize these types of such thermal gradients are known in the prior art, and in general comprise three related approaches.
1. Aluminum-Baclced Pl~te The aluminum-backed plate is described in general molecular biology reference manuals of best practices with no reference to an originator (see Sambrook e~ al., 1988, Molecular Clonine: A Laboratorv Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., vol. 2, pl3.46, a~Zd Ausubel e~ al., 1987, ~0 Current Protocols in Molecular Biolo~v. John Wiley & Sons, N.Y., vol. 1, p7.6.12).
In conventional (usually home-made) ~mbod ~c Or this type Or ele~ p' csis apparatus, an aluminum plate is clamped to the rront or back glass plate containing the gel as a means of evening out the temperature gradient.
However, heat transfer between the glass plate and the aluminum plate is somewhat WO 95/27198 ~ ~ 2 r~ o - ~
variable because of non-uniform flatness of the contacting surfaces and the poorthermal conductivity of air. Using aluminum-backed plate devices, the sidc-to-sidc temperature gradients produced in the gels under conventional conditions of denaturing slab gel electrophoresis are reduced by I to 3C. The "smiling" artifact is reduced substantially using this apparatus, but is nevertheless still detectable and poses a hindrance to easy determination of a nucleotide sequence arrayed within the gel.
2. Buffer-Backed Pi~te The buffer-backed piate type of apparatus (commercially available from Bcthcsda Rcscarch Labs, Gaithcrsburg, MD and BioRad, Hcrculcs, CA) is also dcscribcd in gcncral molccuiar rcfcrcncc manuals of bcst practiccs with no rcfcrcnce to an originator (see Sambrook e~ al., ibid.). The buffcr-backcd platc is somcwhat more effectivc than the aluminum-backed plate because of the more uniform heat transfer bctwccn a frce convcction fluid (buffcr) and thc glass platc. As a rcsult, this systcm rcduccs "smiling" to an acccptablc Icvcl for manual rcading; howcvcr, thc construction of thc scqucncing gcl apparatus is complicatcd by thc rcquircmcnt for a watcr tight sealed compartment that extends the full length of the back platc.
forms the basis for distinguishing a variety of biologically important molccules on the basis of size, charge or a combination thereof. Specific examples of biological molecules advantageously separated by gel electrophoresis include proteins and nucleic aids. Electrophoresis is usually performed in a gellcd (e.g., agarose) or polymeri~ed (e.g., polyacrylamide) media (generically termed a "gel") that contains an electrically conducting buffer. Electrophoresis is performed wherein a voltage ~5 is applied ~ia chemically inert metal electrodes across the cross-sectional arca of the gel. The biological sample of interest is placed into pre-formed sample wells in the I
WO 95/27198 r~.J o l~ -2 1 ~7097 8el. usually at one end of the gel, and the polarity of the applied voltage is arrangcd so that the biological sample migrates through the gel towards one of the electrodes (usually positioned at the opposite end of the gel from the samples). Wherc appropriate, the inverse linear r~ ' ir between migration distance and molecular size is maintained by the addition of chemical denaturants (such as urea, formamide, or sodium dodecyl sulfate) to the gel and cle~,LIu,uh~le~i~ buffer.
A particular application of gel electrophoresis is the separation Or single-stranded DNA fragments in the determination of the nucleotide sequence of a nucleic acid of interest. To this end, a collection of single-stranded DNA fragments is generated either by chemical degradation of the nucleic acid (using the Gilbert method, see e~e" Maxam and Gilbert (1980), Methods Enzyme, ~, p499-5ûO) or by replacement DNA synthesis using a polymerase (using the Sanger method, see e.e.,Sanger, F., Niklen, S., and Coulson, A.R. (1977) Proc. Nat. Acad. Sci. USA 74~ p5463-5467). This co11ection of single stranded DNA fragments includes a fra8ment corresponding to each position in the sequence to be determined; in the most frequently-used sequencing method, this correspondence is directly related to thc distance from a fixed site of initiation of polymerization at a primer that is annealed to the nucleic acid to be sequenced. Thus, determination of the desiredsequence depends on the separation of each of the fragments, which differ in length by only a single nucleotide.
Traditionally, the identity of each of the possible nucleotides at eacb position(adenine, guanine, cytosine or thymidine) is distinguishcd by performing a sequencing reaction specific for each endin8 nucleotide in separate chemical reaction mixtures. Thus, each sequencing experiment is typically perrormcd in 4 separate tubes, wherein are generated a collection of fragmen~s each ending at a W0 95127198 1'~ r ~ --t ~ 2 1 ~370q7 position corresponding to the terminating nucleotide used in that reaction. A
nucleotide sequence is thereafter determined by performing denaturin8 gcl ele~.LIcrho.~is on each of a set of 4 reactions, each reaction electrophoresed individually in adjacent lanes of a single sequencing gel. The presence of a band S at a position in a nucleotide-specific lane of such a gel indicates the identity of that nucleotide at that position in the sequence. Using conventional techniques, each of the fragments is r~tiic~i~b~ , and the bands are visualized after electrophoresis by autoradiography.
Alternatively, recent t~rhnnlogi~ plu~c~cnts have resulted in the development of automated sequencing machines. Such machines contain a spectrophotometric detection means at the distal end of the gel opposite to the loading wells. The DNA fragments are synthesized using either a fluorcscent-labeled primer or fluorescent-labeled nucleotides. In either case, each of thc nucleotide reactions continue to be performed separately, wherein the fragments corresponding to each of the nucleotides is fluorescently labeled with a distinguishable fluorescent label. However, because each fluorescent label exhibits fluorescent emission at a characteristic frequency, it is possible to distinguish thc identity of the nucleotide at the terminal position of each fragment from its fluorescence emission signature. This feature allows the automated collection ornucleotide sequence information spectrophotometrically using these devices. In addition, the existence of distinguishable fluorescent signatures for cach of thc fragments allows the products of the four separate sequencing renctions to be electrophoresed in a single lane of the gel.
These developments have increased the requirements for accuratc and reliable separation of the sequencing fragments generated in nucleotide scquencing WO 95/27198 ,: ~C ~ ' reactions. However, there are a number of technical limitations inherenl in current slab gel elc~,~.uv}.o.cs; technology that have remain unsolved in the current state of this art. For example, limitations on the range of distinguishably-separable fragments necessitates that each sequencing reaction series be loaded onto the gel in two sets of lanes loaded at two separate times. The lanes loaded first (and thus elc~ uvho~c~cdlongest)areusedtodeterminethenucleotidesequencefurthestfrom the polymerization initiation site (and are thus the ~&Ç5~ fragments), while thesecond-loaded set are read to determine the nucleotide sequence closest to the initiation site. The relative timing of loading and the time the reactions are aliowed to elc.,~.uvl-lo~ , are empirically chosen so that these ranges overlap; thus, one entire section of any particular nucleotide sequence can be read. The result of these limits of this CIC~ ù~ Ol~ iS that a maximum of 350-400 bases can be read from a s~ ing, reaction that is initiated at a given site. Thus, the development of methods and apparatuses capable of resolving a longer extent of nucleûtide sequencc than is currently possible would be a useful development in this art.
A number of other technical constraints that limit the extent ûf nucleotide sequence information that can be obtained using conventional electrophoresis techniques are the consequence of the electrophoretic conditions needcd for DNA
sequence analysis using slab gel electrophoresis. Preeminent among these arc thermal temperature gradients produced within the gel during electrophoresis.
In order to separate the single-stranded DNA fragments during electrophoresis proportional to the length of each fragment, a potential Or 1000-3000 volts [(1-3 kilovolts (kV)] is typically applied across the gel for 2-16 hours.
Approximately 30 to 60 watts of power is dissipated uniformly in the gel as heat and then from the surface of the gel plates zontaining the slab gel. Such heating is WO 95/2~198 r~ u~
j~, f;~ 21~9~
useful (and, in fact, nccessary), because it promotes n~oin~rnsnr~ of the single-stranded DNA in the denatured state (so that migration distancc is inverscly proportional to fragment length). However, the extent of such heating is limited to the thermal tolerance of the materials comprising the electrophoresis apparatus.S This limitation places an upper limit on the magnitude of the applied voltage; since the duration of cle~llu~ ,sis depends on the magnitude of the applied voltage, the capacity for decreasing the duration of ele~,l.u,uh~ .is is also constrained by the limitation of the magnitude of the applied power.
An additional problem associated with electrogenic heating of DNA
lû sequencing gels is the uneven distribution of such heat throughout the gel, resulting in thermal temperature gradients in the gel. Under conventional denaturing acrylamide gel ele~ opl.ol~,~is conditions, the central one-quarter to one-third of thc gel becomes warmer during electrophoresis than the rest of the gel throughout its length. Because the mobility of the single stranded DNA fragmcnts varies proportionally with temperature (at an increasing rate of approximately 2.2% perC), the col~s~u,.~,llc~ of uneven heating is that the samples in the center lanes run faster than the samples on the edges. The result is the production of sequencing gels wherein the pattern of bands in the lanes across the gel gives the appearance Or"smiling": the bands are u-o~-~,ii,iv~ily shifted anodally (i.e. upwards using conventional techniques) going from the center of the gel towards the edges.
Another serious problem to which there was no solution proposcd or implemented in the prior art is the problem associated with high-voltagc slab gel electrophoresis -- the gencration of front-to-back thcrmal tcmperature gradients. In particular, the resolution of each of thc bands from all othcr bands rcsolvcd in cach lanc of a sequencing gel is affected by thcsc typcs of thcrmal gradicnts.
WO 95/27198 ~ r P~~ O -r Band resolution depends on the size (i.e., the thickness) of each band, as weil as the relationship between the average thickness of each band and the width of thc space separating each band. A sequencing ladder comprised Of thick bands will contain fewer resoivable bands on average than a gel having thinner, more tightly-resolved bands, simply due tO the finite length of the resolvable portion of the gel.
Also, since each of the bands in a sequeneing gel ean in principle be separ~ated by as few as one nucleotide, stretches Of DNA having multiple repeats Of a particular nucleotide (e.g., 5'-11 1 1 11 1 1 1-3') will be more diffieult (if not impossible) tO
resolve in a gel haYing thicker, less tightly-resolved bands than in a gel having thinner, more tightly-resolved bands. Although this factor is particularly importanl for resolving repetitive stretches Of one type of nucleotide (because the phenomenon isexacerbatedbycl~ opl.o-cslsinthesamelaneofsingle-strandedDNAfragmcnts eorresponding to eaeh of these nucleotides), the problems of band resolution that are band width-related also occur in certain portions Of a gel across more than one gel lane (so-ealled "compression zones").
These problems have been reeognized in the prior art, as is evidenced by the use of [35S]-labeled deoxynucleotides for labeling the DNA in sequencing reactions (see Sambrook e~ al., il7id.). The bandwidths of autoradiographic bands producedfrom ~-partiele emissions from 35S are narrower than the bandwidths produced rrom sZP f-particle emissions beeause ~IsS emits lower-energy particles that convert silver grains over a narrower area in the autoradiographic film. The userulness Of suchnarrower bandwidths is evidenced by the acceptance of this method in the art in thc face of a number of practical disadvantages associated with 35S usc. These includc ~onger autoradiographic development t ~.
~ Wo 95~27198 ~) 1 8 7 ~ ~ 7 . ~ ,a~
A major determinant of the band-width of single-stranded DNA fragments separated by slab gel elecl-opk~ is using high voltages is the generation Or rront-to-back thermal temperature gradients in the gels during high-voltage electrophoresis. Thus, there was an unappreciated and unfulfilled need in the DNA
sequencing/ gel cle~L.o~,h~lc~is art for an apparatus for performing high-voltage, slab gel electrophoresis under conditions in which both front-to-back as well as side-to-side thermal temperature gradients are min' ' Thus, limitations of the prior art include: limits on how high the voltage can be, due to overheating of the gels and thereby resulting in longer run times; limits on ionic strength since higher ionic strengths need higher power; and unevcn heating, causing "smiling" (i.e., left-to-right) distortion and decreased resolution (front-to-back).
Attempts to minimize these types of such thermal gradients are known in the prior art, and in general comprise three related approaches.
1. Aluminum-Baclced Pl~te The aluminum-backed plate is described in general molecular biology reference manuals of best practices with no reference to an originator (see Sambrook e~ al., 1988, Molecular Clonine: A Laboratorv Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., vol. 2, pl3.46, a~Zd Ausubel e~ al., 1987, ~0 Current Protocols in Molecular Biolo~v. John Wiley & Sons, N.Y., vol. 1, p7.6.12).
In conventional (usually home-made) ~mbod ~c Or this type Or ele~ p' csis apparatus, an aluminum plate is clamped to the rront or back glass plate containing the gel as a means of evening out the temperature gradient.
However, heat transfer between the glass plate and the aluminum plate is somewhat WO 95/27198 ~ ~ 2 r~ o - ~
variable because of non-uniform flatness of the contacting surfaces and the poorthermal conductivity of air. Using aluminum-backed plate devices, the sidc-to-sidc temperature gradients produced in the gels under conventional conditions of denaturing slab gel electrophoresis are reduced by I to 3C. The "smiling" artifact is reduced substantially using this apparatus, but is nevertheless still detectable and poses a hindrance to easy determination of a nucleotide sequence arrayed within the gel.
2. Buffer-Backed Pi~te The buffer-backed piate type of apparatus (commercially available from Bcthcsda Rcscarch Labs, Gaithcrsburg, MD and BioRad, Hcrculcs, CA) is also dcscribcd in gcncral molccuiar rcfcrcncc manuals of bcst practiccs with no rcfcrcnce to an originator (see Sambrook e~ al., ibid.). The buffcr-backcd platc is somcwhat more effectivc than the aluminum-backed plate because of the more uniform heat transfer bctwccn a frce convcction fluid (buffcr) and thc glass platc. As a rcsult, this systcm rcduccs "smiling" to an acccptablc Icvcl for manual rcading; howcvcr, thc construction of thc scqucncing gcl apparatus is complicatcd by thc rcquircmcnt for a watcr tight sealed compartment that extends the full length of the back platc.
3. Circulating Wnter Fufther i...p, ~ ,..cnt in the buffer-backcd platc typc of apparatus has bccn obtaincd by using forccd convcction of a~fluid (usually watcr) on the back side of the glass, as described by Garoff and ~O~8C, (1981, Anal. Biochem. 11~: 450) and ~ WO95~27198 IJ , j ~ 2187097 r~
commercially-availablefromPharmacia,Milwaukee,WI. This rmho~1ir~-ntachievcs a very uniform temperature distribution from side to side and yields a pattern Or straight bands in lanes substantially across the width of the gel. This ~mho~linnrnt has been used successfully in conjunction with automated gel sequence readers using stationary photodiode tubes, as described above. However, this embodiment suffers from the disadvantage that a special circulator pump is required this pump supplies both suction and pressure regulated to avoid high pressures inside the jacket which could break the glass-to-plastic water jacket seal during operation. This disadvantageously adds to the cost of such a system. An additional disadvantage is the presence of water in the circulating jacket in proximity to the high voltages associated with DNA sequencing by slab gel ele~ ,pl.o-esis.
commercially-availablefromPharmacia,Milwaukee,WI. This rmho~1ir~-ntachievcs a very uniform temperature distribution from side to side and yields a pattern Or straight bands in lanes substantially across the width of the gel. This ~mho~linnrnt has been used successfully in conjunction with automated gel sequence readers using stationary photodiode tubes, as described above. However, this embodiment suffers from the disadvantage that a special circulator pump is required this pump supplies both suction and pressure regulated to avoid high pressures inside the jacket which could break the glass-to-plastic water jacket seal during operation. This disadvantageously adds to the cost of such a system. An additional disadvantage is the presence of water in the circulating jacket in proximity to the high voltages associated with DNA sequencing by slab gel ele~ ,pl.o-esis.
4. Laminar-flow Air-cooled Cnpillary El~..FI~ t~i~
Weinberger, U.S. Patent No. 5,053,115, issued on October 1, 1991, describes a capillary ele~.opl.~.~Dis apparatus wherein the temperature of the gel is kept constant using a laminar-flow air-cooled jacket or manifold fitted around thc outside of the capillary tube. In this device, a heating element is placed on one sidc of a capillary tube and a fan on the other side, and heat-sensing elements used to maintain a constant temperature across the surface of the capillary tube by laminar air flow.
Owl Scientific, Cambridgc, Massachusetts has marketed a slab gcl electrophoresis device (called thc "Road RunnerTM") that uses the flow of ~o . p. csscd air, such as is commonly found at the benchtop in scicntific laboratories, ovcr slab gcls to cool such gels during electrophoresis by convection. Air warmcd by convective heat transfer from the gel plates is vented from the apparatus to cffcct g wo 9~/27 198 ~ g ~ ` r~l"J,. ' ~
heat withdrawal from the system. Notched aluminum plates are also available to cnhance thc convective cooling provided i~y the apparatus.
~ WO95127198 \i ~ ' 2~37097 r~""..~
SUMMARY OF THE INVENTION
This invention relates to the use of forced air temperature control of gel el~ opk~ a separations. In particular, this invention relates to forced air methods and apparatus for accurately controlling the side-to-side and more illlvol L2lll11y the front-to-back thermal gradients in slab gels to improve the read length of DNA sequences.
In particular, this invention discloses the use of several split flow methods that provide ayllll~ iC~I heat transfer to both sides of the glass gel platcs resulting in lower front-to-back gradients in the gel and a corresponding imp.u~ t in the lû resolution of separated DNA fragments.
A further improvement is obtained using split flow air impingement on thc front and back glass surfaces. The invention is useful in determining DNA
sequences by slab gel elc~lluvho~ and in particular the reading Or DNA
sequences to much longer lengths than is the present practice.
Major contributions of this invention to the art include a number of uniquc ways to achieve balanced temperature gradients in the gel, including: balanced front-to-back temperature gradients in the gel to extend resolution Or separatedDNA fragments; balanced side-to-side temperature gradients in the gel to reducc "smiling" type distortion; and balanced top-to-bottom temperature gradients in thc gel to achieve repeatable run times.
The advantages over prior art apparatus includes the ability to run thc gels at high and uniform temperatures so that the ssDNA remains denatured. Another advantage of the present invention is ability to use low ionic strength bufrers to extend the readability of the runs. The present invention also provides the ability to use higher voltages (and therefore higher power/wattage) to increase the speed W0 95/27198 . P~
2t87097 of extended runs. Stiii another advantage of the present invention is that it provides symmetrical turbulent airflow over the ~el plates, thereby minimizing front-to-back and side-to-side thermal gradients. Other advantages of the present invention include high heat transfer coefficients and the ability to maintain constant thermal conditions at greater or less than ambient temperatures.
The present invention has the ability to decouple the gel temperature from the power dissipation in the gel, allowing optimized conditions.
Wo 95127198 1 ~I/U
2 ~ 8 7~
BRIEF DESCRIPTION OF TE~E DRAWINGS
Figures la through I d illustrate distortions caused by temperature gradients in slab gels after high-voltage el~ opl~ aia~ Figures la and Ic show band patterns using conventional methods and Figures Ib and Id show band patterns using the apparatus of the present invention.
Figures 2a, 2b and 2c represent conventional apparatuses for reducing ~:lule gradients during slab gel electrophoresis.
Figures 3a, 3b and 3c are graphic representations of the temperature profilc of a gel layer between air-cooled glass plates.
Figures 4a and 4b illustrate the extent of thermal contribution to DNA band width.
Figures Sa, Sb and Sc illustrate embodiments of the present invention having forced-air methods for reducing temperature gradients.
Figures 6a and 6b show an ~mbo,'' t of present invention haYing a turbulent-flow,twosidedjetill~,,in~. apparatushavingtwoscrollfanspowered by a single motor drive.
Figures 7a and 7b show another embodiment of the present invention having a double-sided tangential-blower jet impingement system having a single blower.
Figures 8a and 8b show another ~mbo~l I of the present invention having a double-sided tangential-fan system having a single fan at the bottom of thc apparatus.
Figures 9a and 9b show another em~o-iim- nt of the present invcntion having a double-sided air impingement gel temperature control apparatus having a singlcfan at one side of the apparatus.
Figures 10a and 10b show another embodiment of the present invention WO 95/2719~ J~ P~
2 ~ ~70q7 having a single fan at the top of the apparatus.
Figure 11 shows the location of the temperatur~ probc on the back , ~ t plate in the present invention.
Figure 12 is a schematic view of the temperature control system of the S present invention.
~lVO95/27198 2 1 87~q7 P~J~.1.. 5~C~
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DETAILED DESCRIPTION OF THE PREFERRED EMBOi31MENTS
Figures la through Id illustrate distortions caused by temp~rature gradients in slab gels after high-voltage electrophoresis. Figures la and Ic show single-stranded DNA fragment band patterns produced using conYentional methods.
Figures Ib and Id show identical patterns produced using the apparatus accordingto the present invention, i.e. the embodiment shown in Figures 6, 7 and 8.
More specifically, Figure la shows the severe distortion in single-stranded DNA fragment band patterns referred to as "smiling" caused by high side-to-sidc thermal gradients. Figure I b shows the same pattern of DNA fragment bands when such side-to-side thermai gradients are reduced using the present invention.
Figure Ic shows the wider bands caused by spreading due to high front-lo-back thermal gradients. Figure Id shows the thinner band-widths found in gels inwhich front-to-back thermal gradients have been reduced using the present invention. A Co.llpl.isul- of the resoiution of the bands shown in each of Figures Icandldillustratestheshorterextentofnucleotidesequencereadabilitycausedby high front-to-back thermal gradients.
Figures 2a, 2b and 2c represent conventional apparatuses for reducing temperature gradients during slab gel electrophoresis. In Figure 2a, an apparatus I has an aluminum backing plate 2, an upper buffer 3, a lower buffer 4, and a glass gel slab 5. Glass gel slab 5 is comprised of a front glass plate 6 and a back glass plate 7 that are substantially parallel to each other and between them, contain gel 8. In this embodiment, the aluminum backing plate 2 is clamped to the back 9 Or back glass plate 7 to form an int~rface 10.
In Figure 2b, an apparatus 11 has a buffer backed plate 12, an uppcr buffer 13, a lower buffer 14, and a glass gel slab 15. Glass gel slab 15 is compriscd Or a WO95/27198 ~ 2 1 87097 r~ ;c ^-- ~
front glass plate 16 and a back glass plate 17 that are substantially parallel to each other and between them, contain gel 18. In this embodiment, the bufrer backed plate 12 is clamped to the back 19 of back glass plate 17 to form an interface 20.
In Figure 2c, an apparatus 21 has a circulating water plate ~2 having circulating water 23, an upper buffer 24, a lower buffer 25, and a glass gel slab 26.
Glass gel slab 26 is comprised of a front glass plate 27 and a back glass plate 28 that are substantially parallel to each other and between them, contain gel 29. In this hori t, circulating water plate 22 is clamped to the back 30 Or back glass platc28 to form an interface 31. Circulating water 23 enters at entry port 32 and flows through circulating water plate 22 and exits through exit port 33. In this .mh~1im~nt~ circulating water 23 carries away heat from glass gel slab 26 2nd isthen cooled and recirculated by conventional methods which are not shown.
Figures 3a, 3b and 3c are graphic representations of the temperature profilc of a gel layer between air-cooled glass plates. More specifically, Figure 3a shows the cross-section 34 of gel 35 formed between a front glass plate 36 and back glass platc 37 of approximately equal thickness 38; Figure 3b shows the temperature prorile 39 in gel 35 under conditions of symmetrical heat transfer to the surrounding air;
under these ~m-di~ , the temperature gradients in the gel 35 are symmetrical, and this results in the lowest peak parabolic temperature gradient in the gel 35 (~Tgcl 40), and the lowest amount of extra band spreading; Figure 3c shows the temperature profile 41 in the gel 35 under conditions of asymmetrical heat transrcr to the surrounding air; under these conditions, the temperature gradients in the gcl 35 are asymmetrical and more heat is transrerred to one side of the gel 35 than thc other, resulting in a front-to-back thermal temperature gradient (~Tgel 42); this pattern is found in the configurations of gel electrophoresis apparatuses Or thc a, WO95127198 ;~ r ~I I . 21 87(~97 r~l~u. ~o ~-apparatus I having aluminum backed plat~ 2 of Figure 2a, the apparatus 11 havingbuffer backed plate 12 of Figure 2b, and the apparatus 21 having circulating water plate 22 of Figure 2c. Comparison of the ATgel values shown in Figures 3b and 3crcveals a much larger (approximately four-fold) temperature gradient in the gel 35 under asymmetric conditions, resulting in a substantial degradation in band spreading and loss of resolution.
The 200u thin gel is formed between two roughly equal thickness glass plates.
The polyacrylamide gel is typically made with lxTBE buffer and is conductivc.
With several thousand volts applied across the length of the gel, approximately 40 watts of power is uniformly generated in the gel.
If the heat transfer is balanced on both sides of the gel, the tempcraturc gradients are symmetrical resulting in the lowest peak parabolic tempcraturc gradient in the gel, ~Tgel, and the lowest amount of extra band spreading. For typical operating conditions, the gradient in the gel is on the order of 0.3C and the mobility of DNA in the gel has a temperature coefficient of 2.3%/C resulting inband spreading of a fraction of a mm over a 20cm length of the gel.
In the typical case, more heat is conducted toward the aluminum or buffcr backed plate than the other side of the system that is exposed to free air having poor heat transfer. The system in the worst case would have all of the heat conductcd out the back resulting in a much larger temperature gradient in the gel, ~Tgel, and a r~b5t~n~ degradation in band spreading and loss of resolution. . Under these conditions, the temperature gradient in the gel will be four times as great rcsulting in a corresponding loss of resolution of four times.
Figures 4a and 4b illustrate the extent of thermal contribution to DNA band width. Specifically, Figures 4a shows the opposed effects on band-width Or WO 95/27198 ~ ~ r~ o ^ ~
21 ~7097 diffusion 43 and thermal heating 44 for a 100/~ thick gel and a 300~L thick gel relative to the electric filed strength 45 in each gel. This plot shows the expected band width as a function of the electric field (power dissipation in the pel). At very low fields (low power levels), the band width is limited by diffusion of the slow moving DNA fragments in the gel matrix. At higher fields (faster runs) the band width is determined by the parabolic thermal profile in the gel since it causes a non-uniform migration of fragments. Thicker gels have a higher parabolic thermal profile and thus wider band widths for a given field. The trend is to thinncr gels for faster high resolution runs, but thinner gels are more difficult to handle and have lower sample loading capacity.
Figure 4b shows the effects of thermal heating in a 300~-thick gel prepared with IX Tris-borate-EDTA buffer in the best case (i.e., symmetrical heat loss 46) and the worst case (i.e., asymmetrical heat loss 47); the two curves intersect at the point 48 where the electric field strength 45 is about 50 volts/cm.
The solid line shows the thermal spreading effect for a 300u thick gel with a symmetric thermal gradient in the gel (equal heat loss out the front and back surface of the glass plates). If the system is set up such that the heat loss from thc front surface varies, performance is found to track the dashed line. At one point, the heat loss is exactly balanced out the front and back corresponding to where thc dashed lines meet the solid symmetric case line. If the heat flow is not balanced, then one is operating up on one of the dashed lines with radically increascd band widths, Conventional DNA gel fixtures attempt to operate near thc symmetric point by adjusting the hcat losses and power levels. However, this point is not stable and depends on the free convection to the room air, contact rcsistancc of thc back aluminum plate and many other thermal variables that are difficult to predict or - 18 -~ W~951~7198 2 1 87a~7 r~l", ~,1 ., ~
control.
Figures 5a, 5b and 5c illustrate embodiments of the present invention having forced-air methods for reducing temperature gradients. Air flow is shown with arrows. In Figure 5a, turbulent flow one side plate apparatus 49 has an upper buffer 50, a lower buffer 51, a glass gel slab 52, and a fan 53 that is driven by motor 54. Glass gel slab 52 is comprised of a front glass plate 55 and a back glass plate 56 which are s~bs~lnti:~lly parallel to each other and between them, contain gel 57. Fan 53 is positioned so that it faces the back 58 of back glass plate 56. Further, fan 53 has propeller blades 59. Fan 53 circulates air on back 58 of back glass plate 56.
Thus, apparatus 49 is constructed with a large mixed flow imp~ller fan 53 placcdnear the back glass plate 56 and thus, turbulent air is circulated over primarily onc side of glass gel slab 52, i.e., back 58 of back glass plate 56. This approach performs as well as the buffer backed example shown before in controlling side-to-side gradients. No attempt was made to circulate high velocity air on both sides Or the front and back glass plate, and thus the thermal gradients were asymmetric front-to-back and suffered the same resolution losses as the other conventional units.
In Figure 5b, a laminar flow two sides plate apparatus 60 has an upper buffer 61, a lower buffer 62, a glass gel slab 63, and a fan 64 that is driven by motor 65. Glass gel slab 63 is comprised of a front glass plate 66 and a back glass plate 67 which are s-Jbs~n~ ly parallel to each other and between them, contain gel 68. Fan 64 is positioned so that it faces the back 69 of back glass plate 67. Further, fan 64 has blades 70 that extend vertically and are between upper burfer 61 and lower buffer 62. Air is circulated by fan 64 in a laminar flow manner for both back 69of back glass plate 67 and the front 71 of front glass plate 66. This apparatus is WO 95~27198 ~ ~ 3 .3 f , 2 1 ~ 7 9 7 r ~ ., .,.,, c .~ ~
mad~ with a conventional long squirrel-caged blower fan 64 that is mounted to a sidc of the glass gel plates 66 and 67. Fan 64 circulates air and the air is flow split so that ~bs~nti~lly half is passed over the front 71 of front glass plate 66 and2IJhct:-nti:llly half over back 69 of the back glass plate 67, thereby eliminating most of the front-to-back gradients. Front-to-back gradients across the glass plates 66 and 67 measured with thermocouples were 0.3-0.5C nearly five times better than conventional gel ap~ .t~ . A slight side-to-side gradient was present from heat pickup as the air moved across the glass plates.
In Figure 5c, an impingement two sides apparatus 72 has an upper buffer 73, a lower buffer 74, a glass gel slab 75, and a fan 76 that is driven by motor 77. Glass gel slab 75 is comprised of a front glass plate 78 and a back glass plate 79 which arc substantially parallel to each other and between them, contain gel 80. Fan 76 ispositioned below lower buffer 74. Further, fan 76 has propeller blades 81. Furthcr, apparatus 72 also has a front impingemcnt plate 82 that faces front 83 of front glass plate 78, and a back impingement plate 84 that faces back 85 of back glass plate 79.
Both front impingement plate 82 and back impingement plate 84 have impingcment holes 86. Fan 76 circulates air from beneath lower buffer 74 in a split flow manner and up along both front , ;n~ t plate 82 and back ,.~il-i5.,lll-,-~t plate 84 and towards upper buffer 73. The circulating air enters through impingement holes 86and then flows back down towards lower buffer 74. This apparatus having a common fan 76 and split flow and front and back impingement plates respectively facing the front and back glass plates provided better performance than thc prototypes of Figures 5a and 5b. In this apparatus, the air flowing through impingement holes 86 impinge on the glass surfaces, i.c. front 83 and back 85 atsubstantially right angles, thereby creating local turbulence and very high hcat ~ WOgS/27198 ,, ,,, 2 1 8 709 7 transfer coefficients. Apparatus 72 is suh~nti~lly symmetrical in that thc impingement holes 86 line up along both front 83 and back 85 and the air flow isbalanced by having a ~y~ l design and large air distribution chambers.
Apparatus 72 has walls 87 that taper as they extend from the bottom of apparatus72 to the top of apparatus 72. This apparatus reduced the front-to-back thermal gradients across the glass plates to below 0.1C and at the same time provided very low side-to-side gradients of 0.3C. Additional performance is obtained by a s~bst~ntis-lly by li.,.ll construction to maintain symmetrical temperature gradients in the gel 80 itself.
Figures 6a and 6b show another ~-mho~ii t of present invention having a turbulent-flow, two sided jet impingement apparatus 88 having front and back chambers 88' and side chambers 88". Air flow is shown with arrows. Apparatus 88 has an upper buffer 89, a lower buffer 90, a glass gel slab 91, and two scroll fans 92 powered by a single motor drive 93. Glass gel slab 91 is comprised of a front glass plate 92 and a back glass plate 93 which are substantially parallel to each othcr and between them, contain gel 94. Fans 92 are positioned below lower buffer 90.
Further, fans 92 have propeller blades 95. Further, apparatus 88 also has a front impingement plate 96 that faces front 97 of front glass plate 92, and a back illlpi~ t plate 98 that faces back 99 of back glass plate 93. Both front impingement plate 96 and back impingement plate 98 have impingement holes 100.
Air is circulated by fans 92 from below lower buffer 90 and up along and throughchambers 88' and towards upper buffer 89. The circulating air enters through impingement holes 100 and then, as the air moves to sidc chambers 88", the air carries heat away from glass gel slab 91. The air then flows through side chambcrs 88" and to intake 101 of the scroll fans 92. High heat transfer is obtained by the air WO 95/27198 P ~ 2 1 ~3 7 ~ 9 7 ~
flowing at high velocity through the holes 100 in the impingement plates 96 and 98 at sl!bstRn~iRlly right angles to the glass plates 92 and 93.
Figures 7a and 7b show another ~ ' t of the present invention having a double-sided tangential-blower jet impingement apparatus 102 having front and back chambers 102- and side chambers 102". Air flow is shown with arrows.
Apparatus 102 has an upper buffer 103, a lower buffer 104, a glass gel slab 105, and a single blower fan 106 powered by a motor that is not shown. Glass gel slab 105 is comprised of a front glass plate 107 and a back glass plate 108 which are substantially parallel to each other and between them, contain gel 109. Blower fan 106 is positioned below lower buffer 104. Further, apparatus 102 also has a front imrin~ t plate 110 that faces front 11~ of front glass plate 107, and a back impingement plate 112 that faces back 113 of back giass plate 108. Both front ~ p;~ t plate 110 and back impingement plate 112 have impingement holcs (that are not shown but are the same as the impingement holes 100 as shown in Figures 6a and 6b). Air is circulated by blower fan 106 at exit 106' from below lower buffer 104 and up along and through chambers 102' and towards upper buffer103. The circulating air enters through the impingement holes and then, as the air moves to side chambers 102", the air carries heat away from glass gel slab 105. Thc air then flows through side chambers 102" and to the intake 106" of blower fan 106.
High heat transfer is obtained by the air flowing at high velocity through the holcs in the impingement plates 110 and 112 at substantially right angles to thc glassplates 10~ and 108. Apparatus 102 has a barrier 104' that separates the air exiting from exit 106' and thc air returning at intake 106". Apparatus 102 also has tapercd walls 114 having a hinged top lid 114' that can be lifted off to allow access to thc inside of apparatus 102. Apparatus 102 is similar to apparatus 88 shown in Figurcs ~ WO 95/27198 ~ 2 1 ~ 7 0 9 7 r l,u~ o ---6a and 6b in that they both have a tangential double sided apparatus, however they are different in that instead of two scroll fans 92 as in apparatus 88, apparatus 102 has a single tangential blower fan 106.
Figures 8a and 8b show another ~mhori t of the invention having an air illltJ e ~ t gel temperature control apparatus 1 15 having front and back chambers 115'andsidechambersll5". Airflowisshownwitharrows. Apparatusll5hasan upper buffer 116, a lower buffer 117, a glass gel slab 118, and a blower fan 119powered by a single motor 120. Glass gel slab 118 is comprised Or a rront glass plate 121 and a back glass plate 122 which are substantially parallel to each othcr and betweenthem,containgel 123. Blowerfan 119ispositionedbelowlowcrbufrer 117.
Further, blower fan 119 has propeller blades 124. Further, apparatus 115 also has a front illlp ~ t plate 125 that faces front 126 of rront glass plate 121, and aback impingement plate 127 that faces back 128 of back glass plate 122. Both rront il~lpil,g~ lt plate 125 and back impingement plate 127 have impingement holes 129.
Air is circulated by blower fan 119 from b~low lower buffer 117 and up and through chambers 115' and towards upper buffer 116. The circulating air cnters through impingement holes 129 and then, as the air moves to side chambers 1 15", the air carries heat away from glass gel slab 118. The air then flows through sidc chambers 115" and to intake 130 of blower fan 119. High heat transfcr is obtaincd by the air flowing at high velocity through the holes 100 in the impingement plates 125 and 127 at substantially right angles to the g~ass plates 121 and 122. Apparatus 115hasabarrier 117'thatseparatestheairexitingfromexit 124'of blowerfan 119 and the air returning to the blower fan 119 at intake 130. Apparatus 115 also has mounting plates 124" for mounting the motor 120.
Figures 9a and 9b show another embodiment of the invention having an air W095/27~98 ~ 21 8 70 r l,.J. ~o - ~
imrin~l t gel temperature control apparatus 131. Air flow is shown with arrows.
Apparatus 131 has an upper buffer 132, a movable lower buffer 133, a glsss gel slab 134, and a blower fan 135 powered by a single motor 136. Glass gel slab 134 is comprised of a front glass plate 137 and a back glass plate 138 which arc substantially parallel to each other and between them, contain gel 139. Move,able lower buffer 133 can be incrementally moved toward or away from upper buffer 132 in ordcr to ~ ' te various lenpths of glass plates. Figure 9b shows movable lower buffer 133 in two different positions, i.e. position 133' and position 133". In addition, apparatus 131 also has a front impingement plate 140 that faccs front 141 of front glass plate 137, and a back impingement plate 142 that faces back 143 of back glass plate 138. Front i...pillg,. - t plate 140 and back impingcmcnt plate 142 have impingement holes 144. Apparatus 131 also has a diverter plate 145 that is positioned between biower fan 135 and back impingement plate 142. Air iscirculated by blower fan 135 around diverter plate 145 in a split flow manner and IS . around impingement plates 140 and 142. The circulating air enters through i~np- ~ t holes 144 and carries heat away from glass gel slab 134. High heat transfer is obtained by the air flowing at high velocity through the holes 144 in thc impingement plates 140 and 142 at substantially right angles to the glass plates 137 and 138. The air then flows along the glass plates 137 and 138, and then rcturns to the intake 146 of the blower fan 135. Thus, the circulating air enters through pil~ lt holes 144 and then carries heat away from glass gel slab 134 as it flowsback to intake 146 of the blower fan 135. As shown in Figure 9b. the air can return to the blower fan 135 from the bottom and/or the top of apparatus 131. Apparatus131hasabarrierl46'thatseparatestheairexitingfromexitl46"ofblowerfanl35 and the air returning to the blower fan 135 at intake 146.
~ WO95/27198 , ,. 2~ ~7~97 r ~
Figures lOa and lOb show another; ~ t of th~ present invention having an air impingement gel temperature control apparatus 147 having front andback chambers 147' and side chambers 147". Air flow is shown with arrows.
Apparatus 147 has an upper buffer 148, a lower buffer 149, a glass gel slab 150, and S a blower fan ISI. Glass gel slab 150 is comprised of a front glass plate 152 and a back glass plate 153 which are suh~t~nri~llly parallel to each other and betweenthem, contain gel 154. Blower fan 151 is positioned above upper buffer 148.
Further, apparatus 147 has a front impingement plate 155 that faces front 156 offront glass plate 152, and a back impingement plate 157 that faces back 158 of back glass plate 153. Both front illlpillg~ cllt plate ISS and back impingement plate 157 have impingement holes (that are not shown but are the same as the impingement holes 100 as shown in Figures 6a and 6b). Apparatus 147 also has a diverter plate 159 that is positioned around the upper buffer 148. Air is circulated by blowcr fan 151 around the outside surface 160 of diverter plate IS9 and around il~lpi-~c..._..t plates 155 and 157. The circulating air enters through impingement holes and then, as the air moves to sid~ chambers 147", the air carries heat away from glass gcl slab 150. The air then flows through side chambers 147" and to intake 161 of the blower fan 151. High heat transfer is obtained by the air flowing at high velocity through the illl~J ~ t holes in the impingement plates 155 and 157 at substantially right angles to the glass plates 152 and 153. Diverter plate IS9 separates the air exiting from exit 162 of blower fan 151 and the air returning to the blower fan ISI at intake 161.
The foregoing embodiments of the prescnt invention demonstrate that therc are alternati~e positions of the blower fan, sometimes referred to hcrein as a blower or fan, in relation to the rest of the apparatus. Those skilled in thc art will WO 95/27198 i~ r~ s~o ~ r ` ` 21 870~ ~
recognize that the position of the blower fan is a design choice that may involve safety and balancing considerations. However, considering all factors, the hoti t shown in Figures ga and 9b is believed to be the preferred construction.
Figure 11 shows the location of the temporature probe 200 on the back i~pin,3~ t plate 142 in the present invention. Temperature probe 200 can be placed on either the back impingement plates or the front impingement plates of the foregoing ~mho~1im~nts. In the preferred ~-mhort t, temperature probe 200 is placed near the bottom of and on the back impingement plate 142 of Figures 9a and 9b.
Figure 12 is a schematic view of the temperature control system 201 of thc present invention. Temperature control system 201 is a closed loop temperature control system. The Temperature probe 200 is a precision thermistor which is accurate within 0.2C from 0 to 70C. Temperature probe 200 in combination witha fixed resistor 202 results in a voltage divider which converts the resistance changes to voltage variations. Temperature probe 200 is in the high side and thefixed resistor 202 is in the low side of the voltage divider. This makes thc temperature to voltage curve more linear than the temperature probe 200 itself.
The voltage is then converted to a digital signal by a 12-bit analog-to-digital converter ("ADC") 204. The reference voltage 203 supplies the voltage to the voltage divider. This makes the ADC reading ratio-metric. Changes in the referencc voltage will not effect the reading. The error in the reading then consists of: thc tolerance of the temperature probe 200, the tolerance of the resistor 202, the offsct of thc ADC 204, and the linear error of the ADC 204.
The micro-controller 205 reads the ADC 204 once every second. It uscs a look-up table with about 0.1C increments and linear interpolation to calculatc thc Wo95127198 2 ~ ~ 70 9 7 ~ U~
temDerature, The temperature reading is subtracted from the preset value. This is the temperature error. The temperature error is fed to the micro-controlier 205. Thc output controls the heater 207 through pulse width modulation ("PWM"). The micro-controller 205 uses a real time clock at 61 Hz to control the PWM period of I Hz, the heater 207 can be controlled via a solid state relay ("SSR") switch 206 in 1/61 increments.
The heater 207 is powered by the AC line voltage because of the relativel~
high power level of the 450W. The heater 207 can be turned on and of r by the solid state relay switch 206. The solid state relay switch 206 only turns on and of r at thc AC voltage's zero crossing. The frequency of the PWM was selected to be I Hz.
During the I second cycle there will be 120 (100 with 50 Hz) zero erossings Or the AC line voltage. The heater 207 can then be controlled from full off to foll on in 1/120 (or 1/100) increments for finer resolution than the real time clock. The resolution will therefore be 1/61, which is fine enough for the specified control. In the preferred 1- ' t, the temperature is maintained in a range of about 50C
to 60C during gel electrophoresis within a tolerance Or about plus or minus 0.5C.
The roregoing detailed description of the invention has been made in general terms and with respect to several preferred ~hodimrnts, Many of the preferred apparatuses and methods stated herein may be varied by persons skilled in the art without departing from the spirit and scope of the present invention as set forth in the following claims and equivalents.
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Weinberger, U.S. Patent No. 5,053,115, issued on October 1, 1991, describes a capillary ele~.opl.~.~Dis apparatus wherein the temperature of the gel is kept constant using a laminar-flow air-cooled jacket or manifold fitted around thc outside of the capillary tube. In this device, a heating element is placed on one sidc of a capillary tube and a fan on the other side, and heat-sensing elements used to maintain a constant temperature across the surface of the capillary tube by laminar air flow.
Owl Scientific, Cambridgc, Massachusetts has marketed a slab gcl electrophoresis device (called thc "Road RunnerTM") that uses the flow of ~o . p. csscd air, such as is commonly found at the benchtop in scicntific laboratories, ovcr slab gcls to cool such gels during electrophoresis by convection. Air warmcd by convective heat transfer from the gel plates is vented from the apparatus to cffcct g wo 9~/27 198 ~ g ~ ` r~l"J,. ' ~
heat withdrawal from the system. Notched aluminum plates are also available to cnhance thc convective cooling provided i~y the apparatus.
~ WO95127198 \i ~ ' 2~37097 r~""..~
SUMMARY OF THE INVENTION
This invention relates to the use of forced air temperature control of gel el~ opk~ a separations. In particular, this invention relates to forced air methods and apparatus for accurately controlling the side-to-side and more illlvol L2lll11y the front-to-back thermal gradients in slab gels to improve the read length of DNA sequences.
In particular, this invention discloses the use of several split flow methods that provide ayllll~ iC~I heat transfer to both sides of the glass gel platcs resulting in lower front-to-back gradients in the gel and a corresponding imp.u~ t in the lû resolution of separated DNA fragments.
A further improvement is obtained using split flow air impingement on thc front and back glass surfaces. The invention is useful in determining DNA
sequences by slab gel elc~lluvho~ and in particular the reading Or DNA
sequences to much longer lengths than is the present practice.
Major contributions of this invention to the art include a number of uniquc ways to achieve balanced temperature gradients in the gel, including: balanced front-to-back temperature gradients in the gel to extend resolution Or separatedDNA fragments; balanced side-to-side temperature gradients in the gel to reducc "smiling" type distortion; and balanced top-to-bottom temperature gradients in thc gel to achieve repeatable run times.
The advantages over prior art apparatus includes the ability to run thc gels at high and uniform temperatures so that the ssDNA remains denatured. Another advantage of the present invention is ability to use low ionic strength bufrers to extend the readability of the runs. The present invention also provides the ability to use higher voltages (and therefore higher power/wattage) to increase the speed W0 95/27198 . P~
2t87097 of extended runs. Stiii another advantage of the present invention is that it provides symmetrical turbulent airflow over the ~el plates, thereby minimizing front-to-back and side-to-side thermal gradients. Other advantages of the present invention include high heat transfer coefficients and the ability to maintain constant thermal conditions at greater or less than ambient temperatures.
The present invention has the ability to decouple the gel temperature from the power dissipation in the gel, allowing optimized conditions.
Wo 95127198 1 ~I/U
2 ~ 8 7~
BRIEF DESCRIPTION OF TE~E DRAWINGS
Figures la through I d illustrate distortions caused by temperature gradients in slab gels after high-voltage el~ opl~ aia~ Figures la and Ic show band patterns using conventional methods and Figures Ib and Id show band patterns using the apparatus of the present invention.
Figures 2a, 2b and 2c represent conventional apparatuses for reducing ~:lule gradients during slab gel electrophoresis.
Figures 3a, 3b and 3c are graphic representations of the temperature profilc of a gel layer between air-cooled glass plates.
Figures 4a and 4b illustrate the extent of thermal contribution to DNA band width.
Figures Sa, Sb and Sc illustrate embodiments of the present invention having forced-air methods for reducing temperature gradients.
Figures 6a and 6b show an ~mbo,'' t of present invention haYing a turbulent-flow,twosidedjetill~,,in~. apparatushavingtwoscrollfanspowered by a single motor drive.
Figures 7a and 7b show another embodiment of the present invention having a double-sided tangential-blower jet impingement system having a single blower.
Figures 8a and 8b show another ~mbo~l I of the present invention having a double-sided tangential-fan system having a single fan at the bottom of thc apparatus.
Figures 9a and 9b show another em~o-iim- nt of the present invcntion having a double-sided air impingement gel temperature control apparatus having a singlcfan at one side of the apparatus.
Figures 10a and 10b show another embodiment of the present invention WO 95/2719~ J~ P~
2 ~ ~70q7 having a single fan at the top of the apparatus.
Figure 11 shows the location of the temperatur~ probc on the back , ~ t plate in the present invention.
Figure 12 is a schematic view of the temperature control system of the S present invention.
~lVO95/27198 2 1 87~q7 P~J~.1.. 5~C~
,: ,, r .
DETAILED DESCRIPTION OF THE PREFERRED EMBOi31MENTS
Figures la through Id illustrate distortions caused by temp~rature gradients in slab gels after high-voltage electrophoresis. Figures la and Ic show single-stranded DNA fragment band patterns produced using conYentional methods.
Figures Ib and Id show identical patterns produced using the apparatus accordingto the present invention, i.e. the embodiment shown in Figures 6, 7 and 8.
More specifically, Figure la shows the severe distortion in single-stranded DNA fragment band patterns referred to as "smiling" caused by high side-to-sidc thermal gradients. Figure I b shows the same pattern of DNA fragment bands when such side-to-side thermai gradients are reduced using the present invention.
Figure Ic shows the wider bands caused by spreading due to high front-lo-back thermal gradients. Figure Id shows the thinner band-widths found in gels inwhich front-to-back thermal gradients have been reduced using the present invention. A Co.llpl.isul- of the resoiution of the bands shown in each of Figures Icandldillustratestheshorterextentofnucleotidesequencereadabilitycausedby high front-to-back thermal gradients.
Figures 2a, 2b and 2c represent conventional apparatuses for reducing temperature gradients during slab gel electrophoresis. In Figure 2a, an apparatus I has an aluminum backing plate 2, an upper buffer 3, a lower buffer 4, and a glass gel slab 5. Glass gel slab 5 is comprised of a front glass plate 6 and a back glass plate 7 that are substantially parallel to each other and between them, contain gel 8. In this embodiment, the aluminum backing plate 2 is clamped to the back 9 Or back glass plate 7 to form an int~rface 10.
In Figure 2b, an apparatus 11 has a buffer backed plate 12, an uppcr buffer 13, a lower buffer 14, and a glass gel slab 15. Glass gel slab 15 is compriscd Or a WO95/27198 ~ 2 1 87097 r~ ;c ^-- ~
front glass plate 16 and a back glass plate 17 that are substantially parallel to each other and between them, contain gel 18. In this embodiment, the bufrer backed plate 12 is clamped to the back 19 of back glass plate 17 to form an interface 20.
In Figure 2c, an apparatus 21 has a circulating water plate ~2 having circulating water 23, an upper buffer 24, a lower buffer 25, and a glass gel slab 26.
Glass gel slab 26 is comprised of a front glass plate 27 and a back glass plate 28 that are substantially parallel to each other and between them, contain gel 29. In this hori t, circulating water plate 22 is clamped to the back 30 Or back glass platc28 to form an interface 31. Circulating water 23 enters at entry port 32 and flows through circulating water plate 22 and exits through exit port 33. In this .mh~1im~nt~ circulating water 23 carries away heat from glass gel slab 26 2nd isthen cooled and recirculated by conventional methods which are not shown.
Figures 3a, 3b and 3c are graphic representations of the temperature profilc of a gel layer between air-cooled glass plates. More specifically, Figure 3a shows the cross-section 34 of gel 35 formed between a front glass plate 36 and back glass platc 37 of approximately equal thickness 38; Figure 3b shows the temperature prorile 39 in gel 35 under conditions of symmetrical heat transfer to the surrounding air;
under these ~m-di~ , the temperature gradients in the gel 35 are symmetrical, and this results in the lowest peak parabolic temperature gradient in the gel 35 (~Tgcl 40), and the lowest amount of extra band spreading; Figure 3c shows the temperature profile 41 in the gel 35 under conditions of asymmetrical heat transrcr to the surrounding air; under these conditions, the temperature gradients in the gcl 35 are asymmetrical and more heat is transrerred to one side of the gel 35 than thc other, resulting in a front-to-back thermal temperature gradient (~Tgel 42); this pattern is found in the configurations of gel electrophoresis apparatuses Or thc a, WO95127198 ;~ r ~I I . 21 87(~97 r~l~u. ~o ~-apparatus I having aluminum backed plat~ 2 of Figure 2a, the apparatus 11 havingbuffer backed plate 12 of Figure 2b, and the apparatus 21 having circulating water plate 22 of Figure 2c. Comparison of the ATgel values shown in Figures 3b and 3crcveals a much larger (approximately four-fold) temperature gradient in the gel 35 under asymmetric conditions, resulting in a substantial degradation in band spreading and loss of resolution.
The 200u thin gel is formed between two roughly equal thickness glass plates.
The polyacrylamide gel is typically made with lxTBE buffer and is conductivc.
With several thousand volts applied across the length of the gel, approximately 40 watts of power is uniformly generated in the gel.
If the heat transfer is balanced on both sides of the gel, the tempcraturc gradients are symmetrical resulting in the lowest peak parabolic tempcraturc gradient in the gel, ~Tgel, and the lowest amount of extra band spreading. For typical operating conditions, the gradient in the gel is on the order of 0.3C and the mobility of DNA in the gel has a temperature coefficient of 2.3%/C resulting inband spreading of a fraction of a mm over a 20cm length of the gel.
In the typical case, more heat is conducted toward the aluminum or buffcr backed plate than the other side of the system that is exposed to free air having poor heat transfer. The system in the worst case would have all of the heat conductcd out the back resulting in a much larger temperature gradient in the gel, ~Tgel, and a r~b5t~n~ degradation in band spreading and loss of resolution. . Under these conditions, the temperature gradient in the gel will be four times as great rcsulting in a corresponding loss of resolution of four times.
Figures 4a and 4b illustrate the extent of thermal contribution to DNA band width. Specifically, Figures 4a shows the opposed effects on band-width Or WO 95/27198 ~ ~ r~ o ^ ~
21 ~7097 diffusion 43 and thermal heating 44 for a 100/~ thick gel and a 300~L thick gel relative to the electric filed strength 45 in each gel. This plot shows the expected band width as a function of the electric field (power dissipation in the pel). At very low fields (low power levels), the band width is limited by diffusion of the slow moving DNA fragments in the gel matrix. At higher fields (faster runs) the band width is determined by the parabolic thermal profile in the gel since it causes a non-uniform migration of fragments. Thicker gels have a higher parabolic thermal profile and thus wider band widths for a given field. The trend is to thinncr gels for faster high resolution runs, but thinner gels are more difficult to handle and have lower sample loading capacity.
Figure 4b shows the effects of thermal heating in a 300~-thick gel prepared with IX Tris-borate-EDTA buffer in the best case (i.e., symmetrical heat loss 46) and the worst case (i.e., asymmetrical heat loss 47); the two curves intersect at the point 48 where the electric field strength 45 is about 50 volts/cm.
The solid line shows the thermal spreading effect for a 300u thick gel with a symmetric thermal gradient in the gel (equal heat loss out the front and back surface of the glass plates). If the system is set up such that the heat loss from thc front surface varies, performance is found to track the dashed line. At one point, the heat loss is exactly balanced out the front and back corresponding to where thc dashed lines meet the solid symmetric case line. If the heat flow is not balanced, then one is operating up on one of the dashed lines with radically increascd band widths, Conventional DNA gel fixtures attempt to operate near thc symmetric point by adjusting the hcat losses and power levels. However, this point is not stable and depends on the free convection to the room air, contact rcsistancc of thc back aluminum plate and many other thermal variables that are difficult to predict or - 18 -~ W~951~7198 2 1 87a~7 r~l", ~,1 ., ~
control.
Figures 5a, 5b and 5c illustrate embodiments of the present invention having forced-air methods for reducing temperature gradients. Air flow is shown with arrows. In Figure 5a, turbulent flow one side plate apparatus 49 has an upper buffer 50, a lower buffer 51, a glass gel slab 52, and a fan 53 that is driven by motor 54. Glass gel slab 52 is comprised of a front glass plate 55 and a back glass plate 56 which are s~bs~lnti:~lly parallel to each other and between them, contain gel 57. Fan 53 is positioned so that it faces the back 58 of back glass plate 56. Further, fan 53 has propeller blades 59. Fan 53 circulates air on back 58 of back glass plate 56.
Thus, apparatus 49 is constructed with a large mixed flow imp~ller fan 53 placcdnear the back glass plate 56 and thus, turbulent air is circulated over primarily onc side of glass gel slab 52, i.e., back 58 of back glass plate 56. This approach performs as well as the buffer backed example shown before in controlling side-to-side gradients. No attempt was made to circulate high velocity air on both sides Or the front and back glass plate, and thus the thermal gradients were asymmetric front-to-back and suffered the same resolution losses as the other conventional units.
In Figure 5b, a laminar flow two sides plate apparatus 60 has an upper buffer 61, a lower buffer 62, a glass gel slab 63, and a fan 64 that is driven by motor 65. Glass gel slab 63 is comprised of a front glass plate 66 and a back glass plate 67 which are s-Jbs~n~ ly parallel to each other and between them, contain gel 68. Fan 64 is positioned so that it faces the back 69 of back glass plate 67. Further, fan 64 has blades 70 that extend vertically and are between upper burfer 61 and lower buffer 62. Air is circulated by fan 64 in a laminar flow manner for both back 69of back glass plate 67 and the front 71 of front glass plate 66. This apparatus is WO 95~27198 ~ ~ 3 .3 f , 2 1 ~ 7 9 7 r ~ ., .,.,, c .~ ~
mad~ with a conventional long squirrel-caged blower fan 64 that is mounted to a sidc of the glass gel plates 66 and 67. Fan 64 circulates air and the air is flow split so that ~bs~nti~lly half is passed over the front 71 of front glass plate 66 and2IJhct:-nti:llly half over back 69 of the back glass plate 67, thereby eliminating most of the front-to-back gradients. Front-to-back gradients across the glass plates 66 and 67 measured with thermocouples were 0.3-0.5C nearly five times better than conventional gel ap~ .t~ . A slight side-to-side gradient was present from heat pickup as the air moved across the glass plates.
In Figure 5c, an impingement two sides apparatus 72 has an upper buffer 73, a lower buffer 74, a glass gel slab 75, and a fan 76 that is driven by motor 77. Glass gel slab 75 is comprised of a front glass plate 78 and a back glass plate 79 which arc substantially parallel to each other and between them, contain gel 80. Fan 76 ispositioned below lower buffer 74. Further, fan 76 has propeller blades 81. Furthcr, apparatus 72 also has a front impingemcnt plate 82 that faces front 83 of front glass plate 78, and a back impingement plate 84 that faces back 85 of back glass plate 79.
Both front impingement plate 82 and back impingement plate 84 have impingcment holes 86. Fan 76 circulates air from beneath lower buffer 74 in a split flow manner and up along both front , ;n~ t plate 82 and back ,.~il-i5.,lll-,-~t plate 84 and towards upper buffer 73. The circulating air enters through impingement holes 86and then flows back down towards lower buffer 74. This apparatus having a common fan 76 and split flow and front and back impingement plates respectively facing the front and back glass plates provided better performance than thc prototypes of Figures 5a and 5b. In this apparatus, the air flowing through impingement holes 86 impinge on the glass surfaces, i.c. front 83 and back 85 atsubstantially right angles, thereby creating local turbulence and very high hcat ~ WOgS/27198 ,, ,,, 2 1 8 709 7 transfer coefficients. Apparatus 72 is suh~nti~lly symmetrical in that thc impingement holes 86 line up along both front 83 and back 85 and the air flow isbalanced by having a ~y~ l design and large air distribution chambers.
Apparatus 72 has walls 87 that taper as they extend from the bottom of apparatus72 to the top of apparatus 72. This apparatus reduced the front-to-back thermal gradients across the glass plates to below 0.1C and at the same time provided very low side-to-side gradients of 0.3C. Additional performance is obtained by a s~bst~ntis-lly by li.,.ll construction to maintain symmetrical temperature gradients in the gel 80 itself.
Figures 6a and 6b show another ~-mho~ii t of present invention having a turbulent-flow, two sided jet impingement apparatus 88 having front and back chambers 88' and side chambers 88". Air flow is shown with arrows. Apparatus 88 has an upper buffer 89, a lower buffer 90, a glass gel slab 91, and two scroll fans 92 powered by a single motor drive 93. Glass gel slab 91 is comprised of a front glass plate 92 and a back glass plate 93 which are substantially parallel to each othcr and between them, contain gel 94. Fans 92 are positioned below lower buffer 90.
Further, fans 92 have propeller blades 95. Further, apparatus 88 also has a front impingement plate 96 that faces front 97 of front glass plate 92, and a back illlpi~ t plate 98 that faces back 99 of back glass plate 93. Both front impingement plate 96 and back impingement plate 98 have impingement holes 100.
Air is circulated by fans 92 from below lower buffer 90 and up along and throughchambers 88' and towards upper buffer 89. The circulating air enters through impingement holes 100 and then, as the air moves to sidc chambers 88", the air carries heat away from glass gel slab 91. The air then flows through side chambcrs 88" and to intake 101 of the scroll fans 92. High heat transfer is obtained by the air WO 95/27198 P ~ 2 1 ~3 7 ~ 9 7 ~
flowing at high velocity through the holes 100 in the impingement plates 96 and 98 at sl!bstRn~iRlly right angles to the glass plates 92 and 93.
Figures 7a and 7b show another ~ ' t of the present invention having a double-sided tangential-blower jet impingement apparatus 102 having front and back chambers 102- and side chambers 102". Air flow is shown with arrows.
Apparatus 102 has an upper buffer 103, a lower buffer 104, a glass gel slab 105, and a single blower fan 106 powered by a motor that is not shown. Glass gel slab 105 is comprised of a front glass plate 107 and a back glass plate 108 which are substantially parallel to each other and between them, contain gel 109. Blower fan 106 is positioned below lower buffer 104. Further, apparatus 102 also has a front imrin~ t plate 110 that faces front 11~ of front glass plate 107, and a back impingement plate 112 that faces back 113 of back giass plate 108. Both front ~ p;~ t plate 110 and back impingement plate 112 have impingement holcs (that are not shown but are the same as the impingement holes 100 as shown in Figures 6a and 6b). Air is circulated by blower fan 106 at exit 106' from below lower buffer 104 and up along and through chambers 102' and towards upper buffer103. The circulating air enters through the impingement holes and then, as the air moves to side chambers 102", the air carries heat away from glass gel slab 105. Thc air then flows through side chambers 102" and to the intake 106" of blower fan 106.
High heat transfer is obtained by the air flowing at high velocity through the holcs in the impingement plates 110 and 112 at substantially right angles to thc glassplates 10~ and 108. Apparatus 102 has a barrier 104' that separates the air exiting from exit 106' and thc air returning at intake 106". Apparatus 102 also has tapercd walls 114 having a hinged top lid 114' that can be lifted off to allow access to thc inside of apparatus 102. Apparatus 102 is similar to apparatus 88 shown in Figurcs ~ WO 95/27198 ~ 2 1 ~ 7 0 9 7 r l,u~ o ---6a and 6b in that they both have a tangential double sided apparatus, however they are different in that instead of two scroll fans 92 as in apparatus 88, apparatus 102 has a single tangential blower fan 106.
Figures 8a and 8b show another ~mhori t of the invention having an air illltJ e ~ t gel temperature control apparatus 1 15 having front and back chambers 115'andsidechambersll5". Airflowisshownwitharrows. Apparatusll5hasan upper buffer 116, a lower buffer 117, a glass gel slab 118, and a blower fan 119powered by a single motor 120. Glass gel slab 118 is comprised Or a rront glass plate 121 and a back glass plate 122 which are substantially parallel to each othcr and betweenthem,containgel 123. Blowerfan 119ispositionedbelowlowcrbufrer 117.
Further, blower fan 119 has propeller blades 124. Further, apparatus 115 also has a front illlp ~ t plate 125 that faces front 126 of rront glass plate 121, and aback impingement plate 127 that faces back 128 of back glass plate 122. Both rront il~lpil,g~ lt plate 125 and back impingement plate 127 have impingement holes 129.
Air is circulated by blower fan 119 from b~low lower buffer 117 and up and through chambers 115' and towards upper buffer 116. The circulating air cnters through impingement holes 129 and then, as the air moves to side chambers 1 15", the air carries heat away from glass gel slab 118. The air then flows through sidc chambers 115" and to intake 130 of blower fan 119. High heat transfcr is obtaincd by the air flowing at high velocity through the holes 100 in the impingement plates 125 and 127 at substantially right angles to the g~ass plates 121 and 122. Apparatus 115hasabarrier 117'thatseparatestheairexitingfromexit 124'of blowerfan 119 and the air returning to the blower fan 119 at intake 130. Apparatus 115 also has mounting plates 124" for mounting the motor 120.
Figures 9a and 9b show another embodiment of the invention having an air W095/27~98 ~ 21 8 70 r l,.J. ~o - ~
imrin~l t gel temperature control apparatus 131. Air flow is shown with arrows.
Apparatus 131 has an upper buffer 132, a movable lower buffer 133, a glsss gel slab 134, and a blower fan 135 powered by a single motor 136. Glass gel slab 134 is comprised of a front glass plate 137 and a back glass plate 138 which arc substantially parallel to each other and between them, contain gel 139. Move,able lower buffer 133 can be incrementally moved toward or away from upper buffer 132 in ordcr to ~ ' te various lenpths of glass plates. Figure 9b shows movable lower buffer 133 in two different positions, i.e. position 133' and position 133". In addition, apparatus 131 also has a front impingement plate 140 that faccs front 141 of front glass plate 137, and a back impingement plate 142 that faces back 143 of back glass plate 138. Front i...pillg,. - t plate 140 and back impingcmcnt plate 142 have impingement holes 144. Apparatus 131 also has a diverter plate 145 that is positioned between biower fan 135 and back impingement plate 142. Air iscirculated by blower fan 135 around diverter plate 145 in a split flow manner and IS . around impingement plates 140 and 142. The circulating air enters through i~np- ~ t holes 144 and carries heat away from glass gel slab 134. High heat transfer is obtained by the air flowing at high velocity through the holes 144 in thc impingement plates 140 and 142 at substantially right angles to the glass plates 137 and 138. The air then flows along the glass plates 137 and 138, and then rcturns to the intake 146 of the blower fan 135. Thus, the circulating air enters through pil~ lt holes 144 and then carries heat away from glass gel slab 134 as it flowsback to intake 146 of the blower fan 135. As shown in Figure 9b. the air can return to the blower fan 135 from the bottom and/or the top of apparatus 131. Apparatus131hasabarrierl46'thatseparatestheairexitingfromexitl46"ofblowerfanl35 and the air returning to the blower fan 135 at intake 146.
~ WO95/27198 , ,. 2~ ~7~97 r ~
Figures lOa and lOb show another; ~ t of th~ present invention having an air impingement gel temperature control apparatus 147 having front andback chambers 147' and side chambers 147". Air flow is shown with arrows.
Apparatus 147 has an upper buffer 148, a lower buffer 149, a glass gel slab 150, and S a blower fan ISI. Glass gel slab 150 is comprised of a front glass plate 152 and a back glass plate 153 which are suh~t~nri~llly parallel to each other and betweenthem, contain gel 154. Blower fan 151 is positioned above upper buffer 148.
Further, apparatus 147 has a front impingement plate 155 that faces front 156 offront glass plate 152, and a back impingement plate 157 that faces back 158 of back glass plate 153. Both front illlpillg~ cllt plate ISS and back impingement plate 157 have impingement holes (that are not shown but are the same as the impingement holes 100 as shown in Figures 6a and 6b). Apparatus 147 also has a diverter plate 159 that is positioned around the upper buffer 148. Air is circulated by blowcr fan 151 around the outside surface 160 of diverter plate IS9 and around il~lpi-~c..._..t plates 155 and 157. The circulating air enters through impingement holes and then, as the air moves to sid~ chambers 147", the air carries heat away from glass gcl slab 150. The air then flows through side chambers 147" and to intake 161 of the blower fan 151. High heat transfer is obtained by the air flowing at high velocity through the illl~J ~ t holes in the impingement plates 155 and 157 at substantially right angles to the glass plates 152 and 153. Diverter plate IS9 separates the air exiting from exit 162 of blower fan 151 and the air returning to the blower fan ISI at intake 161.
The foregoing embodiments of the prescnt invention demonstrate that therc are alternati~e positions of the blower fan, sometimes referred to hcrein as a blower or fan, in relation to the rest of the apparatus. Those skilled in thc art will WO 95/27198 i~ r~ s~o ~ r ` ` 21 870~ ~
recognize that the position of the blower fan is a design choice that may involve safety and balancing considerations. However, considering all factors, the hoti t shown in Figures ga and 9b is believed to be the preferred construction.
Figure 11 shows the location of the temporature probe 200 on the back i~pin,3~ t plate 142 in the present invention. Temperature probe 200 can be placed on either the back impingement plates or the front impingement plates of the foregoing ~mho~1im~nts. In the preferred ~-mhort t, temperature probe 200 is placed near the bottom of and on the back impingement plate 142 of Figures 9a and 9b.
Figure 12 is a schematic view of the temperature control system 201 of thc present invention. Temperature control system 201 is a closed loop temperature control system. The Temperature probe 200 is a precision thermistor which is accurate within 0.2C from 0 to 70C. Temperature probe 200 in combination witha fixed resistor 202 results in a voltage divider which converts the resistance changes to voltage variations. Temperature probe 200 is in the high side and thefixed resistor 202 is in the low side of the voltage divider. This makes thc temperature to voltage curve more linear than the temperature probe 200 itself.
The voltage is then converted to a digital signal by a 12-bit analog-to-digital converter ("ADC") 204. The reference voltage 203 supplies the voltage to the voltage divider. This makes the ADC reading ratio-metric. Changes in the referencc voltage will not effect the reading. The error in the reading then consists of: thc tolerance of the temperature probe 200, the tolerance of the resistor 202, the offsct of thc ADC 204, and the linear error of the ADC 204.
The micro-controller 205 reads the ADC 204 once every second. It uscs a look-up table with about 0.1C increments and linear interpolation to calculatc thc Wo95127198 2 ~ ~ 70 9 7 ~ U~
temDerature, The temperature reading is subtracted from the preset value. This is the temperature error. The temperature error is fed to the micro-controlier 205. Thc output controls the heater 207 through pulse width modulation ("PWM"). The micro-controller 205 uses a real time clock at 61 Hz to control the PWM period of I Hz, the heater 207 can be controlled via a solid state relay ("SSR") switch 206 in 1/61 increments.
The heater 207 is powered by the AC line voltage because of the relativel~
high power level of the 450W. The heater 207 can be turned on and of r by the solid state relay switch 206. The solid state relay switch 206 only turns on and of r at thc AC voltage's zero crossing. The frequency of the PWM was selected to be I Hz.
During the I second cycle there will be 120 (100 with 50 Hz) zero erossings Or the AC line voltage. The heater 207 can then be controlled from full off to foll on in 1/120 (or 1/100) increments for finer resolution than the real time clock. The resolution will therefore be 1/61, which is fine enough for the specified control. In the preferred 1- ' t, the temperature is maintained in a range of about 50C
to 60C during gel electrophoresis within a tolerance Or about plus or minus 0.5C.
The roregoing detailed description of the invention has been made in general terms and with respect to several preferred ~hodimrnts, Many of the preferred apparatuses and methods stated herein may be varied by persons skilled in the art without departing from the spirit and scope of the present invention as set forth in the following claims and equivalents.
: ! .: ' .
Claims
WHAT IS CLAIMED IS:
1. A gel electrophoresis separation apparatus comprising, in combination:
a gaseous heat-exchange medium, a gaseous heat-exchange medium driving means, and an impingement means, whereby the gaseous heat-exchange medium is driven by the gaseous heat-exchange medium driving means across the impingement means to provide a turbulent flow of the gaseous heat-exchange medium symmetrically across a surface defined by an electrophoretic gel plate, whereby the turbulent flow induced by passage of the gaseous heat-exchange medium through the impingement means thereby minimizes temperature gradients within the gel by convective heat transfer between the heat-exchange medium and the outer surface of the gel plate.
2. The apparatus of Claim 1, wherein the gaseous heat-exchange medium driving means comprises a blower.
3. The apparatus of Claim 1, wherein the gaseous heat-exchange medium is air and the gaseous coolant driving means comprises a fan.
4. A gel electrophoresis separation apparatus according to Claim 1 further comprising:
a temperature control assembly, comprising a volume of a gaseous heat-exchange medium, a gaseous heat-exchange medium containment means arranged to define a gaseous heat-exchange medium volume, a gaseous heat-exchange medium driving means, and an impingement means, whereby the gaseous heat-exchange medium is driven by the gaseous heat-exchange medium driving means, within the gaseous heat-exchange medium containment volume and through the impingement means, causing turbulent flow of the gaseous heat-exchange medium symmetrically across the surface of a gel plate, the turbulent flow induced by passage of the gaseous heat-exchange medium through the impingement means thereby minimizing temperature gradients within the gel byconvective heat transfer between the heat-exchange medium and the outer surface of the gel plate.
5. A gel electrophoresis separation apparatus according to Claim 4, wherein the impingement means comprises a first impingement plate and a second impingement plate;
the impingement plates being approximately equal in size and substantially rectangular, each plate having an inner face and an outer face and a plurality of throughbores; the faces of each impingement plate being oriented approximately parallel to each other; the throughbores disposed approximately perpendicularly to the impingement plate faces and thereby defining impingement passageways;
whereby the first impingement plate is located adjacent to a first gel plate of the gel electrophoresis separation apparatus and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate; and the second impingement plate is located adjacent to a second gel plate of the gel electrophoresis separation apparatus and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate, wherein the first and second gel plates define a gel-containing region located between them.
6. A gel electrophoresis separation apparatus according to Claim 5, wherein the impingement plates and the gel plates are each of approximately equal rectangular cross-sectional area.
7. A gel electrophoresis separation apparatus according to Claim 4, wherein the gaseous heat-exchange medium driving means comprises a blower.
8. A gel electrophoresis separation apparatus according to Claim 4, wherein the gaseous heat-exchange medium is air and the gaseous heat-exchange medium driving means comprises a fan.
9. A gel electrophoresis separation apparatus according to Claim 1 further comprising:
a temperature control assembly, comprising a volume of a gaseous heat-exchange medium, a gaseous heat-exchange medium containment housing arranged to define a gaseous heat-exchange medium volume having an outer periphery defined by a boundary and comprising a first gaseous heat-exchange medium containment region and a second gaseous heat-exchange medium containment region distal to the first region and separated from the first region by a central gaseous heat-exchange medium containment region; means for driving the gaseous heat-exchange medium within the gaseous heat-exchange medium containment volume wherein the gaseous heat-exchange medium driving means is arranged proximally to the second gaseous heat-exchange medium containment region; means for directing the gaseous heat-exchange medium to flow along the outer periphery of the gaseous heat-exchange medium containment volume; and a first and a second impingement plate;
the impingement plates being approximately equal in size and substantially rectangular, each plate having an inner face and an outer face and a plurality of throughbores; the faces of each impingement plate being oriented approximately parallel to each other; the throughbores disposed approximately perpendicularly to the impingement plate faces and thereby defining impingement passageways;
whereby the first impingement plate is located adjacent to a first gel plate of the gel electrophoresis separation apparatus and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate;
the second impingement plate is located adjacent to a second gel plate of the gel electrophoresis separation apparatus and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate, wherein the first and second gel plates define a gel-containing region located between them;
whereby the gaseous heat-exchange medium is driven by the gaseous heat-exchange medium driving means within the gaseous heat-exchange medium containment volume along the periphery towards the first region distal to the driving means, and thereby moving through the central region and being contacted with the outer faces of the impingement plates, whereby the gaseous heat-exchange medium communicates with the plurality of throughbores of each of the impingement plates, causing turbulent flow of the gaseous heat-exchange medium within and into the spaces between the gel plates and the impingement plates, and thereby moving to the second region proximal to the gaseous heat-exchange medium driving means;
the turbulent flow induced by passage of the gaseous heat-exchange medium through the throughbores of the impingement plates thereby minimizing temperature gradients within the gel-containing space between the gel plates by convective heat transfer between the heat-exchange medium and the outer faces of the first and second gel plates.
10. A gel electrophoresis separation apparatus according to Claim 9, wherein the impingement plates and the gel plates are each of approximately equal rectangular cross-sectional area.
11. A gel electrophoresis separation apparatus according to Claim 9, wherein the impingement plates are approximately identical in dimension.
12. A gel electrophoresis separation apparatus according to Claim 9, wherein the gel plates are positioned approximately centrally within the gaseous heat-exchange medium containment housing, thereby bisecting the gaseous heat-exchangemedium containment volume.
13. A gel electrophoresis separation apparatus according to Claim 9, wherein the gaseous heat-exchange medium driving means comprises a blower.
14. A gel electrophoresis separation apparatus according to Claim 9, wherein the means for directing the gaseous heat-exchange medium to flow along the outer periphery of the gaseous heat-exchange medium containment volume comprisesa cap shroud.
15. A gel electrophoresis separation apparatus according to Claim 14, wherein the cap shroud is located in the gaseous heat-exchange medium containment region proximal to the gaseous heat-exchange medium driving means.
16. A gel electrophoresis separation apparatus according to Claim 9, wherein the gaseous heat-exchange medium is air and the gaseous heat-exchange medium driving means comprises a fan.
20. A gel electrophoresis separation apparatus according to Claim 9, wherein the gel plates are positioned approximately centrally within the gaseous heat-exchange medium containment housing, thereby bisecting the gaseous heat-exchangemedium containment volume.
27. A gel electrophoresis separation apparatus according to Claim 9, wherein the gaseous heat-exchange medium containment housing comprising side panels having a length and separated by a distance, wherein the side panels are arranged so that the distance between the side panels increases along the length of the panels, and wherein the arrangement of the side panels subtends an angle between the panels, said angle being from about 10 degrees to about 90 degrees, whereby the cross-sectional area of the volume defined by the containment housing is smaller in the first gaseous heat-exchange medium containment region than in the second gaseous heat-exchange medium containment region.
28. A gel electrophoresis separation apparatus according to Claim 9, wherein the gas heat-exchange medium driving means is oriented substantially perpendicularly to either the first or the second edge of the gel plates.
30. A gel electrophoresis separation apparatus according to Claim 1, wherein gel electrophoresis separation is performed at a temperature greater than ambient temperature.
32. The apparatus of Claim 1 wherein the gaseous heat-exchange medium driving means maintains the temperature of the gaseous heat-exchange medium in arange of about 50°C to 60°C during gel electrophoresis.
33. The apparatus of Claim 32, wherein the gaseous heat-exchange medium driving means maintains the temperature of the gaseous heat-exchange medium within a tolerance range of about plus or minus 0.5°C.
34. A method for maintaining the temperature of an electrophoresis gel on a glass plate during electrophoresis separation comprising:
(a) providing an enclosed chamber which has within it an impingement plate with a plurality of openings, a means for driving a gaseous heat-exchange medium through the openings, and a means for mounting a gel between glass plates in the chamber so that the gaseous heat-exchange medium passing through the openings impinges on the glass plates, (b) placing a gel between glass plates in the chamber on the means for mounting the gel between glass plates, and (c) driving the gaseous heat-exchange medium through the openings in the impingement plate to uniformly heat the electrophoresis gel.
35. The method of Claim 34, wherein the temperature of the gaseous heat-exchange medium is maintained in a range of about 50°C to 60°C during electrophoresis separation.
36. The method of Claim 35, wherein the temperature of the gaseous heat-exchange medium is maintained within a tolerance range of plus or minus 0.5°C.
37. A gel electrophoresis separation apparatus according to Claim 1 further comprising, in combination:
(a) a first gel plate;
(b) a second gel plate;
(c) a first buffer reservoir;
(d) a second buffer reservoir; and (e) a temperature control subassembly;
the first gel plate and the second gel plate being approximately equal in size and substantially rectangular, the gel plates being oriented substantially parallel to one another and defining a gel-containing region, the gel plates each having an inner, gel-contacting face and an outer face, and a first edge, a pair of side edges, and a second edge, wherein the gel-containing region is further defined by a pair of spacers located between the first and second plates substantially along each of the side edges;
the first buffer reservoir being adjacent to the first edge and communicating with the gel-containing region;
the second buffer reservoir being adjacent to the second edge and communicating with the gel-containing region;
the temperature control assembly further having a gaseous heat-exchange medium containment housing arranged to define a gaseous heat-exchange medium volume having an outer periphery defined by a boundary and comprising a first gaseous heat-exchange medium containment region and a second gaseous heat-exchange medium containment region distal to the first region and separated fromthe first region by a central gaseous heat-exchange medium containment region and a volume of a gaseous heat-exchange medium; means for driving the gaseous heat-exchange medium within the gaseous heat-exchange medium containment volume wherein the gaseous heat-exchange medium driving means is arranged proximally tothe second gaseous heat-exchange medium containment region: means for directing the gaseous heat-exchange medium to flow along the outer periphery of the gaseous heat-exchange medium containment volume; and a first impingement plate and a second impingement plate.
wherein the impingement plates are approximately equal in size and substantially rectangular, each plate having an inner face and an outer face and a plurality of throughbores, the faces of each impingement plate being oriented approximately parallel to each other, and the throughbores are disposed approximately perpendicularly to the impingement plate faces and thereby defining impingement passageways;
wherein the first impingement plate is located adjacent to the first gel plate and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate; and the second impingement plate is located adjacent to the second gel plate and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate; whereby the gaseous heat-exchange medium is driven by the gaseous heat-exchange medium driving means within the gaseous heat-exchange medium containment volume along the periphery of the gaseous heat-exchange medium containment housing towards the first region distal to the driving means, and thereby moving through the central region and being contacted with the outer faces of the impingement plates, whereby the gaseous heat-exchange medium communicates with the plurality of throughbores of each of the impingement plates, causing flow of the gaseous heat-exchange medium within and into the spaces between the gel plates and the impingement plates, and thereby moving to the second region proximal to the gaseous heat-exchange medium driving means;
wherein the turbulent flow induced by passage of the gaseous heat-exchange medium through the throughbores of the impingement plates minimizes temperature gradients within the gel-containing space between the gel plates by convective heat transfer between the heat-exchange medium and the outer faces of the first and second gel plates.
38 (37.) A gel electrophoresis separation apparatus according to Claim 40, wherein the gaseous heat-exchange medium driving means comprises a blower.
39. A gel electrophoresis separation apparatus according to Claim 40, wherein the blower is located proximally to either the first or second edge of the gel plates.
40. A gel electrophoresis separation apparatus according to Claim 40, wherein the first and the second buffer reservoirs are physically isolated from the gas heat-exchange medium containment volume.
1. A gel electrophoresis separation apparatus comprising, in combination:
a gaseous heat-exchange medium, a gaseous heat-exchange medium driving means, and an impingement means, whereby the gaseous heat-exchange medium is driven by the gaseous heat-exchange medium driving means across the impingement means to provide a turbulent flow of the gaseous heat-exchange medium symmetrically across a surface defined by an electrophoretic gel plate, whereby the turbulent flow induced by passage of the gaseous heat-exchange medium through the impingement means thereby minimizes temperature gradients within the gel by convective heat transfer between the heat-exchange medium and the outer surface of the gel plate.
2. The apparatus of Claim 1, wherein the gaseous heat-exchange medium driving means comprises a blower.
3. The apparatus of Claim 1, wherein the gaseous heat-exchange medium is air and the gaseous coolant driving means comprises a fan.
4. A gel electrophoresis separation apparatus according to Claim 1 further comprising:
a temperature control assembly, comprising a volume of a gaseous heat-exchange medium, a gaseous heat-exchange medium containment means arranged to define a gaseous heat-exchange medium volume, a gaseous heat-exchange medium driving means, and an impingement means, whereby the gaseous heat-exchange medium is driven by the gaseous heat-exchange medium driving means, within the gaseous heat-exchange medium containment volume and through the impingement means, causing turbulent flow of the gaseous heat-exchange medium symmetrically across the surface of a gel plate, the turbulent flow induced by passage of the gaseous heat-exchange medium through the impingement means thereby minimizing temperature gradients within the gel byconvective heat transfer between the heat-exchange medium and the outer surface of the gel plate.
5. A gel electrophoresis separation apparatus according to Claim 4, wherein the impingement means comprises a first impingement plate and a second impingement plate;
the impingement plates being approximately equal in size and substantially rectangular, each plate having an inner face and an outer face and a plurality of throughbores; the faces of each impingement plate being oriented approximately parallel to each other; the throughbores disposed approximately perpendicularly to the impingement plate faces and thereby defining impingement passageways;
whereby the first impingement plate is located adjacent to a first gel plate of the gel electrophoresis separation apparatus and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate; and the second impingement plate is located adjacent to a second gel plate of the gel electrophoresis separation apparatus and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate, wherein the first and second gel plates define a gel-containing region located between them.
6. A gel electrophoresis separation apparatus according to Claim 5, wherein the impingement plates and the gel plates are each of approximately equal rectangular cross-sectional area.
7. A gel electrophoresis separation apparatus according to Claim 4, wherein the gaseous heat-exchange medium driving means comprises a blower.
8. A gel electrophoresis separation apparatus according to Claim 4, wherein the gaseous heat-exchange medium is air and the gaseous heat-exchange medium driving means comprises a fan.
9. A gel electrophoresis separation apparatus according to Claim 1 further comprising:
a temperature control assembly, comprising a volume of a gaseous heat-exchange medium, a gaseous heat-exchange medium containment housing arranged to define a gaseous heat-exchange medium volume having an outer periphery defined by a boundary and comprising a first gaseous heat-exchange medium containment region and a second gaseous heat-exchange medium containment region distal to the first region and separated from the first region by a central gaseous heat-exchange medium containment region; means for driving the gaseous heat-exchange medium within the gaseous heat-exchange medium containment volume wherein the gaseous heat-exchange medium driving means is arranged proximally to the second gaseous heat-exchange medium containment region; means for directing the gaseous heat-exchange medium to flow along the outer periphery of the gaseous heat-exchange medium containment volume; and a first and a second impingement plate;
the impingement plates being approximately equal in size and substantially rectangular, each plate having an inner face and an outer face and a plurality of throughbores; the faces of each impingement plate being oriented approximately parallel to each other; the throughbores disposed approximately perpendicularly to the impingement plate faces and thereby defining impingement passageways;
whereby the first impingement plate is located adjacent to a first gel plate of the gel electrophoresis separation apparatus and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate;
the second impingement plate is located adjacent to a second gel plate of the gel electrophoresis separation apparatus and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate, wherein the first and second gel plates define a gel-containing region located between them;
whereby the gaseous heat-exchange medium is driven by the gaseous heat-exchange medium driving means within the gaseous heat-exchange medium containment volume along the periphery towards the first region distal to the driving means, and thereby moving through the central region and being contacted with the outer faces of the impingement plates, whereby the gaseous heat-exchange medium communicates with the plurality of throughbores of each of the impingement plates, causing turbulent flow of the gaseous heat-exchange medium within and into the spaces between the gel plates and the impingement plates, and thereby moving to the second region proximal to the gaseous heat-exchange medium driving means;
the turbulent flow induced by passage of the gaseous heat-exchange medium through the throughbores of the impingement plates thereby minimizing temperature gradients within the gel-containing space between the gel plates by convective heat transfer between the heat-exchange medium and the outer faces of the first and second gel plates.
10. A gel electrophoresis separation apparatus according to Claim 9, wherein the impingement plates and the gel plates are each of approximately equal rectangular cross-sectional area.
11. A gel electrophoresis separation apparatus according to Claim 9, wherein the impingement plates are approximately identical in dimension.
12. A gel electrophoresis separation apparatus according to Claim 9, wherein the gel plates are positioned approximately centrally within the gaseous heat-exchange medium containment housing, thereby bisecting the gaseous heat-exchangemedium containment volume.
13. A gel electrophoresis separation apparatus according to Claim 9, wherein the gaseous heat-exchange medium driving means comprises a blower.
14. A gel electrophoresis separation apparatus according to Claim 9, wherein the means for directing the gaseous heat-exchange medium to flow along the outer periphery of the gaseous heat-exchange medium containment volume comprisesa cap shroud.
15. A gel electrophoresis separation apparatus according to Claim 14, wherein the cap shroud is located in the gaseous heat-exchange medium containment region proximal to the gaseous heat-exchange medium driving means.
16. A gel electrophoresis separation apparatus according to Claim 9, wherein the gaseous heat-exchange medium is air and the gaseous heat-exchange medium driving means comprises a fan.
20. A gel electrophoresis separation apparatus according to Claim 9, wherein the gel plates are positioned approximately centrally within the gaseous heat-exchange medium containment housing, thereby bisecting the gaseous heat-exchangemedium containment volume.
27. A gel electrophoresis separation apparatus according to Claim 9, wherein the gaseous heat-exchange medium containment housing comprising side panels having a length and separated by a distance, wherein the side panels are arranged so that the distance between the side panels increases along the length of the panels, and wherein the arrangement of the side panels subtends an angle between the panels, said angle being from about 10 degrees to about 90 degrees, whereby the cross-sectional area of the volume defined by the containment housing is smaller in the first gaseous heat-exchange medium containment region than in the second gaseous heat-exchange medium containment region.
28. A gel electrophoresis separation apparatus according to Claim 9, wherein the gas heat-exchange medium driving means is oriented substantially perpendicularly to either the first or the second edge of the gel plates.
30. A gel electrophoresis separation apparatus according to Claim 1, wherein gel electrophoresis separation is performed at a temperature greater than ambient temperature.
32. The apparatus of Claim 1 wherein the gaseous heat-exchange medium driving means maintains the temperature of the gaseous heat-exchange medium in arange of about 50°C to 60°C during gel electrophoresis.
33. The apparatus of Claim 32, wherein the gaseous heat-exchange medium driving means maintains the temperature of the gaseous heat-exchange medium within a tolerance range of about plus or minus 0.5°C.
34. A method for maintaining the temperature of an electrophoresis gel on a glass plate during electrophoresis separation comprising:
(a) providing an enclosed chamber which has within it an impingement plate with a plurality of openings, a means for driving a gaseous heat-exchange medium through the openings, and a means for mounting a gel between glass plates in the chamber so that the gaseous heat-exchange medium passing through the openings impinges on the glass plates, (b) placing a gel between glass plates in the chamber on the means for mounting the gel between glass plates, and (c) driving the gaseous heat-exchange medium through the openings in the impingement plate to uniformly heat the electrophoresis gel.
35. The method of Claim 34, wherein the temperature of the gaseous heat-exchange medium is maintained in a range of about 50°C to 60°C during electrophoresis separation.
36. The method of Claim 35, wherein the temperature of the gaseous heat-exchange medium is maintained within a tolerance range of plus or minus 0.5°C.
37. A gel electrophoresis separation apparatus according to Claim 1 further comprising, in combination:
(a) a first gel plate;
(b) a second gel plate;
(c) a first buffer reservoir;
(d) a second buffer reservoir; and (e) a temperature control subassembly;
the first gel plate and the second gel plate being approximately equal in size and substantially rectangular, the gel plates being oriented substantially parallel to one another and defining a gel-containing region, the gel plates each having an inner, gel-contacting face and an outer face, and a first edge, a pair of side edges, and a second edge, wherein the gel-containing region is further defined by a pair of spacers located between the first and second plates substantially along each of the side edges;
the first buffer reservoir being adjacent to the first edge and communicating with the gel-containing region;
the second buffer reservoir being adjacent to the second edge and communicating with the gel-containing region;
the temperature control assembly further having a gaseous heat-exchange medium containment housing arranged to define a gaseous heat-exchange medium volume having an outer periphery defined by a boundary and comprising a first gaseous heat-exchange medium containment region and a second gaseous heat-exchange medium containment region distal to the first region and separated fromthe first region by a central gaseous heat-exchange medium containment region and a volume of a gaseous heat-exchange medium; means for driving the gaseous heat-exchange medium within the gaseous heat-exchange medium containment volume wherein the gaseous heat-exchange medium driving means is arranged proximally tothe second gaseous heat-exchange medium containment region: means for directing the gaseous heat-exchange medium to flow along the outer periphery of the gaseous heat-exchange medium containment volume; and a first impingement plate and a second impingement plate.
wherein the impingement plates are approximately equal in size and substantially rectangular, each plate having an inner face and an outer face and a plurality of throughbores, the faces of each impingement plate being oriented approximately parallel to each other, and the throughbores are disposed approximately perpendicularly to the impingement plate faces and thereby defining impingement passageways;
wherein the first impingement plate is located adjacent to the first gel plate and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate; and the second impingement plate is located adjacent to the second gel plate and arranged to substantially cover it, and defining a space between the gel plate and the impingement plate; whereby the gaseous heat-exchange medium is driven by the gaseous heat-exchange medium driving means within the gaseous heat-exchange medium containment volume along the periphery of the gaseous heat-exchange medium containment housing towards the first region distal to the driving means, and thereby moving through the central region and being contacted with the outer faces of the impingement plates, whereby the gaseous heat-exchange medium communicates with the plurality of throughbores of each of the impingement plates, causing flow of the gaseous heat-exchange medium within and into the spaces between the gel plates and the impingement plates, and thereby moving to the second region proximal to the gaseous heat-exchange medium driving means;
wherein the turbulent flow induced by passage of the gaseous heat-exchange medium through the throughbores of the impingement plates minimizes temperature gradients within the gel-containing space between the gel plates by convective heat transfer between the heat-exchange medium and the outer faces of the first and second gel plates.
38 (37.) A gel electrophoresis separation apparatus according to Claim 40, wherein the gaseous heat-exchange medium driving means comprises a blower.
39. A gel electrophoresis separation apparatus according to Claim 40, wherein the blower is located proximally to either the first or second edge of the gel plates.
40. A gel electrophoresis separation apparatus according to Claim 40, wherein the first and the second buffer reservoirs are physically isolated from the gas heat-exchange medium containment volume.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US22258394A | 1994-04-04 | 1994-04-04 | |
| US08/222,583 | 1994-04-04 | ||
| US35257794A | 1994-12-09 | 1994-12-09 | |
| US08/352,577 | 1994-12-09 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2187097A1 true CA2187097A1 (en) | 1995-10-12 |
Family
ID=26916962
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2187097 Abandoned CA2187097A1 (en) | 1994-04-04 | 1995-04-03 | Control of temperature gradients during gel electrophoresis using turbulent coolant gas flow |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0777856A1 (en) |
| JP (1) | JPH10502446A (en) |
| CN (1) | CN1150476A (en) |
| AU (1) | AU2276795A (en) |
| CA (1) | CA2187097A1 (en) |
| WO (1) | WO1995027198A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5942432A (en) * | 1997-10-07 | 1999-08-24 | The Perkin-Elmer Corporation | Apparatus for a fluid impingement thermal cycler |
| WO2001020317A1 (en) * | 1999-09-13 | 2001-03-22 | Riken | Method for preparing supporter for electrophoresis |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CS171472B1 (en) * | 1974-03-04 | 1976-10-29 | ||
| FR2653560B2 (en) * | 1988-03-02 | 1994-09-02 | Helena Lab Corp | AUTOMATIC ELECTROPHORESIS METHOD AND APPARATUS. |
| US5053115A (en) * | 1990-01-25 | 1991-10-01 | Spectra-Physics, Inc. | Automated neutral marker for capillary electrophoresis |
| US5122253A (en) * | 1990-05-11 | 1992-06-16 | Hewlett-Packard Company | Transverse forced gas cooling for capillary zone electrophoresis |
-
1995
- 1995-04-03 WO PCT/US1995/004065 patent/WO1995027198A1/en not_active Application Discontinuation
- 1995-04-03 JP JP7525888A patent/JPH10502446A/en active Pending
- 1995-04-03 CA CA 2187097 patent/CA2187097A1/en not_active Abandoned
- 1995-04-03 EP EP95916173A patent/EP0777856A1/en not_active Withdrawn
- 1995-04-03 AU AU22767/95A patent/AU2276795A/en not_active Abandoned
- 1995-04-03 CN CN 95193409 patent/CN1150476A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CN1150476A (en) | 1997-05-21 |
| AU2276795A (en) | 1995-10-23 |
| WO1995027198A1 (en) | 1995-10-12 |
| JPH10502446A (en) | 1998-03-03 |
| EP0777856A1 (en) | 1997-06-11 |
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