WO2005114158A1

WO2005114158A1 - Multiple detection electrophoresis

Info

Publication number: WO2005114158A1
Application number: PCT/US2005/016649
Authority: WO
Inventors: Zhaowei Liu; Thomas E. Kane
Original assignee: Spectrumedix Llc
Priority date: 2004-05-14
Filing date: 2005-05-13
Publication date: 2005-12-01

Abstract

In a method for separating components of a sample, the sample is subjected to electrophoresis along a separation lane while subjecting components of the sample to at least a first temperature. Without removing the sample from the separation lane, components of the sample that have been subjected to the first temperature are detected. Then, without removing the sample from the separation lane, components of the sample are subjected to electrophoresis along the separation lane while subjecting the sample to a least a second temperature. Components of the sample that have been subjected to the second temperature are detected. The first and second temperatures are different.

Description

MULTIPLE DETECTION ELECTROPHORESIS

RELATED APPLICATIONS The present application claims the benefit of U.S. provisional application no. 60/570,831, filed May 14, 2004, which application is incorporated herein by reference.

FIELD OF THE JJNVENTION The present invention relates to methods and devices for electrophoretic separation of sample components.

BACKGROUND OF THE INVENTION Temperature gradient electrophoresis (TGE), e.g., temperature gradient capillary electrophoresis (TCGE), includes subjecting a sample to a temperature gradient to induce partial melting of heteroduplex DNA components as compared to homoduplex components. Determining the appropriate temperature (or temperature gradient) to separate heteroduplexes and homoduplexes may require more than one electrophoresis run. Thus, temperature gradient electrophoresis analyses often require multiple runs of the same sample.

SUMMARY OF THE INVENTION One aspect of the present invention relates to a method for separating components of a sample. In one embodiment, the sample is subjected to electrophoresis along a separation lane while subjecting components of the sample to at least a first temperature. Without removing the sample from the separation lane, components of the sample that have been subjected to the first temperature are detected. Without removing the sample from the separation lane, components of the sample are subjected to electrophoresis along the separation lane while subjecting the sample to a least a second temperature. Components of the sample that have been subjected to the second temperature are detected. The first and second temperatures are typically different. In some embodiments, components of the sample are subjected to the first temperature within a first portion of the separation lane and components of the sample are subjected to the second temperature within a second, different portion of the separation lane. The temperature within the first portion and/or second portions may be essentially constant, e.g., during the time a given sample component takes to migrate through the portion. For example, the temperature may vary, e.g., during the time a given sample component takes to migrate through the portion, by 5 °C or less, 2.5 °C or less, 1 °C or less, e.g., 0.5 °C or less. In some embodiments, the temperature within the first portion is essentially constant, e.g., during the time a given sample component takes to migrate through the portion, and the temperature within the second portion varies by more than 5 °C, 7.5 °C or more, e.g., 10 °C or more, e.g., during the time it takes the given sample component and/or another sample component to migrate through the second portion. The essentially constant temperature (or the average thereof) within the first portion may be less than the average temperature within the second portion, e.g., at least 5 °C less, at least 7.5 °C less, at least 10 °C less, at least 15 °C less, e.g., at least 20 °C less. Another aspect of the present invention relates to a system for separating components of a sample. In one embodiment, the system includes, a separation lane defining a separation axis. The separation lane may include, an inlet, and a detection zone, spaced apart by a distance Dl along the separation axis. The separation lane generally includes first and second portions consecutively disposed along the separation axis. The first portion has a length ΔL1 along the separation axis. The second portion has a length ΔL2 along the separation axis. The first and second portions are located intermediate the inlet and the detection zone. A ratio ΔL1/D1 may be at least 0.1, at least 0.15, at least 0.2, at least 0.25, e.g., at least 0.3. The ratio ΔL1/D1 may be less than 0.7, less than 0.5, less than 0.4, e.g., about 0.3 or less. A ratio ΔL2/D1 may be at least 0.1, at least 0.15, at least 0.2, at least 0.25, e.g., at least 0.3. The ratio ΔL2/D1 may be less than 0.7, less than 0.5, less than 0.4, e.g., about 0.3 or less. The system may include a thermal controller configured to simultaneously and/or independently subject the first portion of the separation lane to a first temperature and the second portion of the separation lane to a second, different temperature. The first and second temperatures may be varied temporally and/or spatially during electrophoresis. In some embodiments, the respective temperatures within the first and second portions is varied temporally but is spatially uniform. In some embodiments, the temperature within the first portion and/or second portions is essentially constant, e.g., during the time a given sample component takes to migrate through the portion. For example, the temperature may vary, e.g., during the time a given sample component takes to migrate through the portion, by 5 °C or less, 2.5 °C or less, 1 °C or less, e.g., 0.5 °C or less. In some embodiments, the temperature within the first portion is essentially constant, e.g., during the time a given sample component takes to migrate through the portion, and the temperature within the second portion varies by more than 5 °C, 7.5 °C or more, e.g., 10 °C or more, e.g., during the time it takes the given sample component and/or another sample component to migrate through the second portion. The essentially constant temperature (or the average thereof) within the first portion may be less than the average temperature within the second portion, e.g., at least 5 °C less, at least 7.5 °C less, at least 10 °C less, at least 15 °C less, e.g., at least 20 °C less. In some embodiments, the detection zone is a first detection zone and the first and second portions are spaced apart by a second detection zone. The temperature within either or both of the first and second detection zones may be less than the average temperature within either or both of the first and second portions when sample components are migrating therethrough. In some embodiments, the temperature within either or both of the first and second detection zones is 30 °C or less, 25 °C or less, 22.5 °C or less, 20 °C or less, e.g., 17.5 °C or less. System components may advantageously be integrated, e.g., a single detection system may be used to detect sample components at each of a plurality of detection zones. A single set of electrophoresis data may be obtained, with the set of electrophoresis data including data from more than one detection zone. Reagents and sample are conserved by detecting the sample more than once during an electrophoresis run. In some embodiments, sample passes through a first temperature gradient before being detected a first time and a second temperature gradient before being detected a second time. In some embodiments, sample passes through a constant temperature region before being detected a first time and a temperature gradient before being detected a second time. The temperature prior to the first detection may be substantially less than the melting point of duplexes to be detected. For example, the temperature may. be less than about 30 °C, e.g., about 25 °C or less. Data from the first detection may be used as a quality control. Should additional loops would provide greater resolution of the data through a larger temperature ramp.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a prior art electrophoresis system. Fig. 2 is a prior art electrophoresis system. Fig. 3 is an electrophoresis system with two detection zones. Fig. 4 is an electrophoresis system having two detection zones and two thermal regions. The system is configured to subject sample components within each thermal region to different temperatures and or gradients. Fig. 5 is another electrophoresis system with two detection zones and two thermal regions. Fig. 6 is a capillary of the system of Fig. 5. Fig. 7 is a capillary having multiple detection zones. Fig. 8 is a plurality of capillaries each having a plurality of detection zones. Fig. 9a is an electrophoresis system with two thermal regions. Fig. 9b shows the status that single stranded DNA and double stranded DNA would assume when migrating within the thermal regions of the system of Fig. 9a. Figs. 9c, 9d, and 9e show electrophoresis data that may be obtained using the system of Fig. 9a. Figs. 10a and 10b show exemplary temperature gradients that may be applied using the electrophoresis systems of Figs. 4, 5 or 9a.

DETAILED DESCRIPTION A method for separating components of a sample includes subjecting a sample to electrophoresis along a separation lane. While subjecting the sample to electrophoresis, components of the sample are subjected to at least a first temperature, e.g., as part of a temperature gradient applied to the sample components. After subjecting the sample to electrophoresis and without removing the sample from the separation lane, components of the sample that have been subjected to the first temperature are detected at a first detection zone. Then, without removing the sample from the separation lane, components of the sample are subjected to further electrophoresis along the separation lane. While subjecting the sample to further electrophoresis, components of the sample are subjected to a second, different temperature, e.g., as part of a different temperature gradient. Components of the sample that have been subjected to the second temperature are detected at a second detection zone. In general, samples to be separated include a plurality of components. In some embodiments, the first temperature typically introduces a migration velocity difference between some but not all components of the sample. The migration velocity difference causes the sample components with the migration velocity difference to separate from one another. Sample components separated by application of the first temperature are detected at the first detection zone. The second temperature introduces a migration velocity difference between other components of the sample. The migration velocity difference causes these sample components to separate from one another. Sample components separated by application of the second temperature are detected at the second detection zone. In some embodiments a first sample component includes a first set of polynucleotides and a second sample component includes a second set of polynucleotides. Each set of polynucleotides includes one or more pairs of polynucleotides, e.g., PCR products. Each pair of polynucleotides includes first and second single polynucleotide strands. The single polynucleotide strands of each pair are sufficiently complementary to form a duplex, e.g., a double stranded DNA molecule. Thus, at sufficiently low temperatures, each set of polynucleotides may include one or more duplexes. Typically, the polynucleotides of different sets of polynucleotides have lengths that differ by an amount sufficient to allow the polynucleotides of one set of polynucleotides to be separated from the polynucleotides of another set. In some embodiments, the duplexes of a given set of polynucleotides have different melting points. For example, the duplexes of a set of polynucleotides may be identical except for the presence or absence of a mismatch. The presence of the mismatch decreases the melting point of a duplex as compared to a duplex missing the mismatch. Duplexes that are otherwise identical except for the presence or absence of a mismatch generally migrate at substantially the same velocity during electrophoresis. In the absence of a migration velocity difference, compounds generally cannot be separated by electrophoresis. When the duplexes are subjected to a temperature intermediate the melting points of the two duplexes, the duplexes migrate at different velocities. Thus, one may determine whether a sample component includes two or more pairs of duplexes differing by the presence or absence of a mismatch by subjecting the duplexes to such a temperature during electrophoresis and determining whether the duplexes have separated. For example, the temperature may be sufficient to at least partially melt one of the duplexes but insufficient to melt the other duplex. Alternatively, the temperature may be sufficient to completely melt one of the duplexes and just sufficient to partially melt the other duplex. In general, the duplexes are subjected to a first temperature gradient encompassing the first temperature and a second temperature gradient encompassing the second temperature to ensure that each respective pair of duplexes is subjected to a temperature sufficient to modify the respective mobility of the pair. Detection of duplexes is typically accomplished optically, using a light source and fluorescence detector. The duplexes migrate within a medium including a fluorescent tag, such as an intercalating dye. Intercalating dyes, e.g., ethidium bromide, fluoresce when intercalated with a duplex but exhibit substantially reduced or no fluorescence when not intercalated. Referring to Fig. 1, an electrophoresis system 100 includes a separation lane, e.g., a capillary 102 having an inlet 104, a detection zone 106, and a detection system, e.g., an optical detection system 108, which detects sample components and provides electrophoresis data 110 indicative of the presence of the sample components. Optical detection system 108 typically includes a light source, e.g., a laser, focusing optics to direct a laser beam from the laser to a detection zone, detection optics to collect fluorescence from the detection zone and a detector to detect the fluorescence. In some embodiments, the detector is an imaging detector configured to simultaneously detect fluorescence from more than one spaced apart detection zone. The detector may include dispersing elements to disperse different wavelengths of the fluorescence from one or more detection zones onto different portions of the detector. Capillary 102 defines an internal bore therealong. The internal bore is generally filled with an electrophoresis medium, e.g., a gel, a polymer, or other matrix, suitable for separating polynucleotides. During electrophoresis, components of the sample migrate within the internal bore generally along a separation axis of the capillary. A distance along the separation axis between the inlet 104 and the detection zone 106 is i. As can be seen in Fig. 1, electrophoresis data 110 includes an unresolved peak 112 and a pair of resolved peaks 114a, 114b. One could not determine from peak 112 alone whether the peak was indicative of the presence of more than one (e.g., two or more) sample components.. Referring to Fig. 2, an electrophoresis system 100' is identical to system 100 except that samples migrate a longer distance before detection. System 100' includes a capillary 102' having an inlet 104' and a detection zone 106'. Optical detection system 108 of system 100' detects sample components and provides electrophoresis data 110'. Capillary 102' defines an internal bore along. During electrophoresis, components of the sample migrate within the internal bore of capillary 102' generally along a separation axis of the capillary 102' . A distance along the separation axis between the inlet 104' and the detection zone 106' is d₂, where d₂ > di. Electrophoresis data 110' and electrophoresis data 110 are obtained from samples having the same composition and size. Because capillary 102' is longer than capillary 102, electrophoresis data obtained using systems 100 and 100' exhibit different resolutions. For example, as seen in Fig. 2, electrophoresis data 110' contains two sets of at least partially resolved peaks. A first set of peaks includes peaks 112a', 112b', and 112c' corresponds to the single overlapped peak 112 of data 110. The presence of multiple peaks 112a', 112b', and 112c' indicates the presence of multiple sample components. Peaks 114a' and 114b' of data 110' are not as well resolved as peaks 114a and 114b of data 110 even though these two sets of peaks correspond to the same sample constituents. Referring to Fig. 3, an electrophoresis system 100" is identical to system 100' except that capillary 102" includes a second detection zone 106a and a second optical detection system 108', which detects the presence of sample components at detection zone 106a. A distance between inlet 104' and detection zone 106a along the separation axis of capillary 102' ' is the same as distance di and a distance between inlet 104' and detection zone 106' along the separation axis of capillary 102" is the same as distance d₂. Sample components may migrate between inlet 104' and detection zone 106' without being removed from capillary 102' . Thus, system 100' ' may be used to obtain electrophoresis data 110 and electrophoresis data 110' without removing sample components from capillary 102" . Referring to Fig. 4, an electrophoresis system 200 is identical with system 100" except that system 200 further includes a thermal controller 220 having a first portion 222 and a second portion 224. First portion 222 subjects sample components to at least a first temperature when the components are migrating between inlet 104' and detection zone 106a. Second portion 224 subjects sample components to at least a second temperature when the constituents are migrating between detection zone 106a and detection zone 106'. An exemplary operation of system 200 is discussed with reference to a sample including a first set of duplexes and a second set of duplexes. Each set of duplexes includes at least two duplexes, e.g., each set of duplexes includes a pair of duplexes. System 200 may be configured so that the temperature within the first portion 222 is intermediate the melting points of one pair of duplexes and the temperature within the second portion 224 is intermediate the melting points of another pair of duplexes of the sample. For example, the temperature within first portion 222 may be sufficient to at least partially melt, e.g., at least partially denature, one duplex of the first pair of duplexes but insufficient to melt the second duplex of the first pair. In general, the temperature within portion 222 is insufficient to completely melt both duplexes of the first pair of duplexes. The temperature within the second portion 224 is generally sufficient to at least partially melt, e.g., at least partially denature, one duplex of the second pair of duplexes but insufficient to melt the second duplex of the second pair. In general, the temperature at detection zone 106' is insufficient to completely melt both duplexes of the second pair of duplexes. Electrophoresis data 210a obtained at detection zone 106a include an overlapped peak 212 and first and second resolved peaks 214a and 214b. Electrophoresis data 210b obtained at detection zone 106b include resolved peaks 212a, 212b, and 212c and an area 214' containing no peaks. Peaks 214a and 214b correspond to the presence of first and second duplexes differing by the presence or absence of a mismatch. Prior to reaching detection zone 106a, these duplexes are subjected to a temperature intermediate their respective melting points causing the first and second duplexes to separate from one another. Peaks 212a, 212b, and 212c correspond to the presence of third, fourth, and fifth duplexes differing, e.g., by the presence or absence of a mismatch. Prior to reaching detection zone 106a, these duplexes are not subjected to a temperature intermediate any of their respective melting points for a time sufficient to allow their separation. Consequently, the third, fourth, and fifth duplexes remain overlapped at detection zone 106a as evidenced by peak 212. Area 214' of electrophoresis data corresponds to the time at which polynucleotides of the first and second duplexes reached detection zone 106'. However, the temperature within portion 224 was sufficiently high to completely melt the first and second duplexes causing the fluorescence of the intercalating dye to decrease. Referring to Fig. 5, an electrophoresis system 300 includes a separation lane, e.g., a capillary 302 having an inlet 304 and first and second detection zones 306a, 306b. Sample components migrate within capillary 302 along a separation axis thereof. System 300 also includes an optical detection system 308. Electrophoresis system 300 is identical with electrophoresis system 200 except that capillary 302 is looped so that detection regions 306a and 306b are separated by a lateral distance that is small (Fig. 6) compared the distance separating the detection regions along the separation axis of capillary 302. Thus, optical detection system 308 can be used to detect sample components at both detection zone 306a and detection zone 306b. For example, the same collection optic, e.g., a lens, may be used to collect light from both detection zones and image the light simultaneously onto the same detector. System 300 may include a thermal controller to subject a first portion of capillary 302 between inlet 304 and detection zone 306a to a first temperature and/or first gradient and to subject a second portion of capillary 302 between detection zone 306a and detection zone 306b to a second temperature and/or second gradient. Referring to Fig. 7, a separation lane, e.g., a capillary 402, may be configured with a plurality of detection zones 306. Capillary 402 has in internal bore, which defines a separation axis. During electrophoresis, sample components migrate generally along the separation axis. In the configuration shown, a minimum distance d₃ between successive detection zones 406 is less than a distance d₄ between the successive detection zones along the separation axis, i.e., the distance traveled by sample components between successive detection zones along the bore of capillary 402. In some embodiments, the ratio of d₃/d₄ is 0.25 or less, 0.1 or less, 0.05 or less, 0.025 or less, e.g., 0.01 or less. In general, portions of capillary 402 intermediate successive detection zones may be subjected to different temperatures. For example, prior to being detected a first time, sample components may be subjected to a first temperature, which may be essentially constant, and/or a temperature gradient. Intermediate the first detection and prior to be detected a second time, sample components may be subjected to a second, different temperature, and/or gradient. More generally, intermediate the N^Λ detection and prior to the N^Λ + 1 detection, sample components may be subjected to an N^Λ typically different temperature, which may be essentially constant, and/or temperature gradient. Referring to Fig. 8, a plurality of separation lanes, e.g., a plurality of capillaries 502, are arranged so that fluorescence may be detected simultaneously from one or more detection zones 506 of each capillary. Another aspect of the invention relates to a method for increasing the detection efficiency, i.e., the detection probability, of DNA mutations. A sample including both single stranded DNA and double stranded DNA is subjected to electrophoresis along a single separation lane, e.g., a single capillary, to cause single stranded DNA molecules to separate from one another and double stranded DNA molecules to separate from one another. In general, the metnoα comDines smgie-srranαeu (ss) DNA temperature gradient capillary electrophoresis (TGCE) and double-stranded (duplex) DNA TGCE. The overall detection efficiency is given by 1 - (1 - α)(l - β), where α is the mutation detection probability for single stranded DNA TGCE and β is the mutation detection probability for double stranded DNA TGCE. In general, 1 - (1 - α)(l - β) > α or β when 0 < α, β < 1. Thus, the use of both single stranded and double stranded TGCE may improve detection efficiencies over the use of either technique alone. Typically, PCR products are subjected to electrophoresis with a temperature profile and/or a buffer system having an ionic strength sufficient to generate roughly similar amounts of double stranded DNA (homo- and heterozygous), single stranded forward DNA, and single stranded reverse DNA. The proportion of single stranded DNA to double stranded DNA in solution is determined by the temperature profile used and the ionic strength of the buffer. For example, PCR products may be diluted in a 10 mM Tris-HCl (pH 8.5) buffer to achieve approximately equal amounts of single stranded DNA and double stranded DNA. In this regard, a paper by Kourkine IV, Hestekin CN, Buchholz BA and Barron AE, Analytical Chemistry, 74:2565-2572, 2002, titled High-Throughput, High-Sensitivity Genetic Mutation Detection By Tandem Single- Strand Conformation Polymorphism/Heteroduplex Analysis Capillary Array Electrophoresis, is attached and incorporated by reference herein. PCR products in an appropriate buffer are injected into a separation lane, e.g. a capillary, and separated by electrophoresis. Typically, the PCR products are fluorescently labeled and/or an intercalating dye may be used. During separation, the sample may be subjected to two different temperature gradients. In one temperature gradient, the temperature is configured to cause different single DNA strands present in the sample to assume different conformations and migrate at different velocities. For example, the maximum temperature is generally less than about 40 °C, less than about 35 °C, e.g., less than about 30 °C. For example, a temperature gradient of from about 20 to about 24 °C or from about 26 to about 30 °C may be used. Alternative to first temperature gradient, the sample components may be subjected to an essentially constant temperature. During a second temperature gradient, the temperature is raised by an amount sufficient to at least partially denature at least some of the double stranded DNA present in the sample. The temperature during the second temperature gradient typically reaches at least 30 °C, at least 40 °C, at least 50 °C, e.g., at least 60 °C. During the second temperature gradient, single stranded DNA may unfold thereby minimizing conformational differences between different strands. As discussed above, however, such temperature gradients are sufficient to separate different double stranded DNA molecules, e.g., different duplexes. All separated polynucleotides, whether single stranded DNA or double stranded DNA may be detected when passing through the detection window and recorded as separated fluorescent signals. The DNA may be detected after both the first and second temperature gradients. In general, the presence of a mutation is determined if either single stranded DNA TGCE or double stranded DNA TGCE indicates a mutation pattern (heterozygous mutation called by both double stranded DNA TGCE and single stranded DNA TGCE, or a homozygous mutation called by single stranded DNA TGCE). Thus, the method is able to determine the presence of genotypes AA, AG, and GG for a diploid organism in a single electrophoresis run. A molecular ladder can be used as a ruler to calibrate the migration variation among different capillaries. The pattern of relative distance between different peaks in either homozygous mutant or heterozygous mutant should also be useful for data analysis when comparing with the pattern of the known wild-type or heterozygous mutation control. Referring to Fig. 9a, an electrophoresis system 600 includes first and second separation lanes for separating components of a sample and a control. In a first portion 602 of the system 600, compounds are subjected to a first temperature, which may be an essentially constant temperature or a temperature gradient. In a second portion 604 of system 600 compounds are subjected to a second temperature gradient. System 600 includes first and second detectors 633,635. First detector 633 detects sample components after being subjected to the first temperature and, generally, before being subjected to the second temperature. Second detector 635 detects sample components after being subjected to the second temperature. Referring to Fig. 9b, the status of DNA during separation within system 600 is shown. Within portion 602, like sized double stranded DNA migrates at substantially the same velocity while single stranded DNA assumes different conformations and, therefore, different migration velocities. Within portion 604, single stranded DNA migrates at the same velocity while melting point differences amount double stranded DNA causes the different double stranded DNA's to migrate at different velocities. Referring to Figs. 9a, 9b, and 9c, the presence or absence of mutations and the genotype of the sample may be determined based upon the peaks present in electrophoresis data. Referring to Figs. 10a and 10b, the temperature during the second temperature gradient is typically higher than that during the first temperature gradient. It should be understood, however, that system 600 may be configured so that the temperature during the first temperature gradient is higher than the temperature during the second temperature gradient. Any of the electrophoresis systems discussed herein may be used to subject a single sample to consecutive single stranded TGCE and double stranded TGCE.

Claims

What is claimed is: 1. A method for separating components of a sample, the method comprising: subjecting a sample to electrophoresis along a separation lane while subjecting components of the sample to at least a first temperature; without removing the sample from the separation lane, detecting components of the sample that have been subjected to the first temperature; then, without removing the sample from the separation lane, subjecting components of the sample to electrophoresis along the separation lane while subjecting the sample to a least a second temperature; and without removing the sample from the separation lane, detecting components of the sample that have been subjected to the second temperature.

2. The method of claim 1, wherein the sample comprises at least first, second, third, and fourth pairs of complementary polynucleotide strands, and further wherein: one of the first and second temperatures is intermediate the melting point of a duplex comprising the first pair of complementary polynucleotide strands and the melting point of a duplex comprising the second pair of complementary polynucleotide strands; and the other of the first and second temperatures is intermediate the melting point of a duplex comprising the third pair of complementary polynucleotide strands and the melting point of a duplex comprising the fourth pair of complementary polynucleotide strands.

3. The method of claim 2, wherein the first and second temperatures differ by at least about 10 °C.

4. The method of claim 2, wherein the first temperature is essentially constant and the second temperature varies by more than 5 °C.

5. The method of claim 1, wherein the separation lane is defined by a capillary.

6. The method of claim 5, comprising: subjecting a second sample to electrophoresis along a second, different separation lane while subjecting components of the second sample to at least a third temperature; without removing the second sample from the second separation lane, detecting components of the second sample that have been subjected to the third temperature; then, without removing the second sample from the second separation lane, subjecting components of the second sample to electrophoresis along the second separation lane while subjecting the second sample to a least a fourth temperature; and detecting components of the second sample that have been subjected to the fourth temperature.

7. The method of claim 6, wherein the second, different separation lane is a second capillary.

8. The method of claim 7, wherein detecting components of the sample that have been subjected to the first temperature comprises irradiating a first portion of the capillary and detecting components of the second sample that have been subjected to the third temperature comprises irradiating a second portion of the second capillary, wherein the first portion of the capillary and the second portion of the second capillary are spaced apart by a minimum distance of less than 5% of a length of the capillary.

9. The method of claim 7, wherein the first and third temperatures are essentially identical and the second and fourth temperatures are essentially identical.

10. A method for separating components of a sample, the method comprising: subjecting a sample to electrophoresis for a distance ΔLj_. along a separation lane having a length L, the sample having a plurality of components, L being greater than ΔLj_.; while subjecting the sample to electrophoresis over the distance ΔLi, subjecting the sample to a temperature Tj; detecting at least one component of the sample that has migrated the distance ΔLt ; then, without removing the sample from the separation lane, subjecting the sample to electrophoresis for a distance ΔL₂ along the separation lane, L being at least as great as ΔLi + ΔL^; while subjecting the sample to electrophoresis over the distance ΔL , subjecting the sample to a temperature T₂, T₂ being more than 5 °C greater than Ti.

11. The method of claim 10, wherein Ti is essentially constant and T₂ varies by at least 5 °C.

12. A device for separating components of a sample, the device comprising: a separation lane defining a separation axis, the separation lane comprising: an inlet; a detection zone, the inlet and detection zone being spaced apart by a distance Dj_. along the separation axis; first and second portions consecutively disposed along the separation axis, the first portion having a length ΔLi along the separation axis, the second portion having a length ΔL₂ along the separation axis, the first and second portions being located intermediate the inlet and the detection zone, wherein a ratio ΔL1/D1 is at least

0.25 and a ratio ΔL₂/Dι is at least 0.25; and a thermal controller configured to simultaneously subject the first portion of the separation lane to a first temperature and the second portion of the separation lane to a second, different temperature.

13. The device of claim 12, wherein the detection zone is a first detection zone and the separation lane comprises a second detection zone disposed intermediate the first and second portions of the separation lane.

14. The device of claim 13, wherein the first and second detection zones are spaced apart by a distance D₂ along the separation axis, and wherein a shortest distance D₃ between the first and second detection zones is less than 10% of D₂.

15. The device of claim 13, wherein the separation lane has a maximum lateral dimension of less than about 250 microns.

16. The device of claim 13, wherein the separation lane is a capillary.

17. The device of claim 13, wherein the separation lane is defined by a substantially planar substrate.