Apparatus and method for the generation, separation, detection, and recognition of biopolymer fragments

• US 6,017,434 A
• Filed: 05/09/1995
• Issued: 01/25/2000
• Est. Priority Date: 05/09/1995
• Status: Expired due to Fees

• Current Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Original Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Inventors: Went, Gregory T., Simpson, John W., Rothberg, Jonathan Marc
• Primary Examiner(s): McKane, Elizabeth
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US08/438,231
• Time in Patent Office: 1,722 Days
• Field of Search: 204/451, 204/452, 204/453, 204/454, 204/455, 204/601, 204/602, 204/603, 204/604, 204/456, 204/457, 204/458, 204/459, 204/461, 204/462, 204/463, 204/464, 204/465, 204/466 , 204/467, 204/468, 204/469, 204/470, 204/606, 204/607, 204/608, 204/609, 204/610, 204/612, 204/613, 204/614, 204/615, 204/616, 435/287.2, 435/287.1, 356/344
• US Class Current: 204/612
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00783 Laminate assemblies, i.e. the reactor comprising a stack of plates
• CPC Class Codes: B01J 2219/00828 Silicon wafers or plates
• CPC Class Codes: B01J 2219/00831 Glass
• CPC Class Codes: B01J 2219/00833 Plastic
• CPC Class Codes: B01J 2219/00873 Heat exchange
• CPC Class Codes: B01J 2219/00995 Mathematical modeling
• CPC Class Codes: G01N 27/44708 Cooling
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44782 of a plurality of samples
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention is an integrated instrument for the high-capacity electrophoretic analysis of biopolymer samples. It comprises a specialized high-voltage, electrophoretic module in which the migration lanes are formed between a bottom plate and a plurality of etched grooves in a top plate, the module permitting concurrent separation of 80 or more separate samples. In thermal contact with the bottom plate is a thermal control module incorporating a plurality of Peltier heat transfer devices for the control of temperature and gradients in the electrophoretic medium. Fragments are detected by a transmission imaging spectrograph which simultaneously spatially focuses and spectrally resolves the detection region of all the migration lanes. The spectrograph comprises a transmission dispersion element and a CCD array to detect signals. Signal analysis comprises the steps of noise filtering, comparison in a configuration space with signal prototypes, and selection of the best prototype. Optionally post-processing is done by a Monte-Carlo simulated annealing algorithm to improve results. Optionally, an array of micro-reactors can be integrated into the instrument for the generation of sequencing reaction fragments directly from crude DNA samples.

Claims

1. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium;

(b) an electrophoresis module for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium, said electrophoresis module comprising

(i) a substantially flat bottom plate; and

(ii) a substantially flat top plate having a plurality of grooves on one side, the top plate being positioned in contact with the bottom plate so that the grooves form with the bottom plate a plurality of separated channels, the channels holding the electrophoretic medium to form electrophoretic migration lanes;

(c) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples; and

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components and generation of output signals representative thereof, said means for substantially simultaneous resolutions being a transmission imaging spectrograph comprising

(i) an optic assembly positioned to receive a substantial fraction of the plurality of light emissions for simultaneous spatial imaging along a first axis and spectral dispersion along a second axis, and

(ii) a detector array for simultaneous spatial and spectral detection of the plurality of light emissions imaged along the first axis and dispersed along the second axis by the optic assembly, the detector producing output signals representative of the detected light;

wherein the grooves in the top plate are spaced to converge toward one end of the plate.

2. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium;

(b) an electrophoresis module for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium, said electrophoresis module comprising

(i) a substantially flat bottom plate; and

(ii) a substantially flat top plate having a plurality of grooves on one side, the top plate being positioned in contact with the bottom plate so that the grooves form with the bottom plate a plurality of separated channels, the channels holding the electrophoretic medium to form electrophoretic migration lanes;

(c) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples;

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components and generation of output signals representative thereof, said means for substantially simultaneous resolution being a transmission imaging spectrograph comprising

(i) an optic assembly positioned to receive a substantial fraction of the plurality of light emissions for simultaneous spatial imaging along a first axis and spectral dispersion along a second axis, and

(ii) a detector array for simultaneous spatial and spectral detection of the plurality of light emissions imaged along the first axis and dispersed along the second axis by the optic assembly, the detector producing output signals representative of the detected light;

(e) cross-lane grooves between selected adjacent lanes forming cross-lane connecting channels, the channels holding separation medium so that biopolymer fragments can migrate between the selected adjacent lanes through the channels; and

(f) electrodes formed in the walls of the selected lanes adjacent to the cross-lane connecting channels for causing fragment migration through the connecting channels upon being energized with voltage.

3. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium;

(b) an electrophoresis module for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium, said electrophoresis module comprising

(i) a substantially flat bottom plate; and

(ii) a substantially flat top plate having a plurality of grooves on one side, the top plate being positioned in contact with the bottom plate so that the grooves form with the bottom plate a plurality of separated channels, the channels holding the electrophoretic medium to form electrophoretic migration lanes;

(c) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples; and

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components and generation of output signals representative thereof, said means for substantially simultaneous resolution being a transmission imaging spectrograph comprising

(i) an optic assembly positioned to receive a substantial fraction of the plurality of light emissions for simultaneous spatial imaging along a first axis and spectral dispersion along a second axis, and

(ii) a detector array for simultaneous spatial and spectral detection of the plurality of light emissions imaged along the first axis and dispersed along the second axis by the optic assembly, the detector producing output signals representative of the detected light;

wherein the top plate is a thermal conductor coated with an electrical insulator, and the grooves are formed on the insulator.

4. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium;

(b) means for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium;

(c) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples; and

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components and generation of output signals representative thereof, said means for substantially simultaneous resolution being a transmission imagine spectrograph comprising

(i) an optic assembly positioned to receive a substantial fraction of the plurality of light emissions for simultaneous spatial imaging along a first axis and spectral dispersion along a second axis, and

(ii) a detector array for simultaneous spatial and spectral detection of the plurality of light emissions imaged along the first axis and dispersed along the second axis by the optic assembly, the detector producing output signals representative of the detected light;

wherein the separation medium comprises small spheres of polystyrene.

5. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium;

(b) means for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium;

(c) means for substantially simultaneously stimulating, light emissions from fragments in a plurality of biopolymer fragment samples; and

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components and generation of output signals representative thereof, said means for substantially simultaneous resolution being a transmission imaging spectrograph comprising

(i) an optic assembly positioned to receive a substantial fraction of the plurality of light emissions for simultaneous spatial imaging along a first axis and spectral dispersion along a second axis, and

(ii) a detector array for simultaneous spatial and spectral detection of the plurality of light emissions imaged along the first axis and dispersed along the second axis by the optic assembly, the detector producing output signals representative of the detected light;

wherein the optic assembly further comprises;

(1) a collection lens positioned to initially receive and collimate the plurality of light emissions;

(2) a transmission dispersion element for spectrally dispersing the collimated signals along the first axis; and

(3) a focusing lens for spatially focusing the signals along the second axis onto the detector array.

6. The apparatus of claim 5 further comprising a spectral filter element positioned between the collection lens and transmission dispersion element for filtering from the light emissions extraneous light.

7. The apparatus of claim 5 in which the transmission dispersion element is a transmission diffraction grating, or a transmission grating-prism.

8. An apparatus for the concurrent analysis of a plurality of DNA sequencing reaction fragment samples, each sample comprising a plurality of labeled DNA sequencing reaction fragments generated from one DNA sample, the labels being dyes with distinctive spectral characteristics, the apparatus comprising:

(a) means for loading on the apparatus the plurality of DNA fragment samples for separation;

(b) an electrophoresis module for the substantially concurrent separation of each of the plurality of DNA fragment samples, the electrophoresis module further comprising;

i. a substantially flat bottom plate; and

ii. a substantially flat top plate having a plurality of grooves on one side, the top plate being positioned in contact with the bottom plate so that the grooves form with the bottom plate a plurality of separated channels, the channels holding the electrophoretic medium to form electrophoretic migration lanes;

(c) a heat control subunit for maintaining a controlled uniform temperature in the electrophoresis module, the subunit further comprising;

i. a heat sink for exchanging heat with the surroundings;

ii. a plurality of thermal transfer devices disposed between and in thermal contact with the heat sink and the bottom plate for bi-directional heat transfer;

(d) a laser illuminating the separated biopolymer fragment samples to stimulate emission of the dye labels and thereby to generate the plurality of light signals;

(e) a transmission imaging spectrograph for substantially simultaneous detection of each of the plurality of light signals, the spectrograph further comprising;

i. an optic assembly positioned to receive a substantial fraction of the plurality of light signals for simultaneous spatial imaging along a first axis and spectral dispersion along a second axis; and

ii. a detector array for simultaneous spatial and spectral detection of the plurality of light signals imaged along the first axis and dispersed along the second axis by the optic assembly, the detector producing output signals representative of the detected light;

(f) means for the analysis of the detection output signals to determine the sequences of the DNA samples from the detector comprising;

i. memory means for storing output signal data, processed signal data, and prototype signal data;

ii. means for cumulating the detection output signals into spatial samples representative of one migration lane, and for each spatial sample, for cumulating the detection output signals into spectral samples representative of the distinctive spectral characteristics of the dye labels;

iii. means for comparing the time behavior of the spectral samples with a set of prototype time behaviors also stored in memory and for selecting prototypes that most closely match the spectral samples; and

iv. means for outputting identities of the selected prototypes as the DNA sequences of the DNA samples.

9. The apparatus of claim 8 wherein the apparatus further comprises:

(a) means for trimming from the output identities of the DNA samples known DNA sequences in the DNA sample;

(b) means for proofreading in a Monte Carlo manner the trimmed DNA sequences, the means for proofreading comprising repetitively making at a random point in the trimmed output a random sequence alteration and evaluating sequence improvement until no further substantial sequence improvement occurs; and

(c) means for outputting the improved sequence as the DNA sequences of the DNA samples.

10. An integrated apparatus for concurrent separation and detection of a plurality of biopolymer fragment samples, each said sample comprising a plurality of biopolymer fragments, the apparatus comprising:

(a) an electrophoresis module for the substantially concurrent electrophoretic separation of said biopolymer fragments within each of said plurality of biopolymer fragment samples, said separation occurring along migration lanes in said electrophoresis module;

(b) means for substantially simultaneously stimulating light emissions from said biopolymer fragments when present in said migration lanes;

(c) an optic assembly comprising a transmission dispersion element selected from the group consisting of a transmission grating-prism and a transmission diffraction grating, said optic assembly for substantially simultaneous spatially imagine and spectrally dispersing said light emissions from said biopolymer fragments onto a detector array, said spatial imaging being along a first axis, and said spectral dispersing being into rays of differing wavelength and along a second axis; and

(d) said detector array for producing output signals representative of said spatially imaged and spectrally dispersed light emissions;

wherein said optic assembly further comprises;

(i) a collection lens for receiving and collimating said light emissions before spectral dispersion by said transmission dispersion element; and

(ii) a focusing lens for focusing said light emissions onto said detector array after spectral dispersion by said transmission dispersion element.

11. The apparatus of claim 10 wherein said collection lens and said focusing lens demagnify said light emissions onto said detector array.

12. The apparatus of claim 10 further comprising a spectral filter element for filtering extraneous light from said light emissions.

13. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each said sample comprising a plurality of biopolymer fragments, the apparatus comprising:

(a) an electrophoresis module for the substantially concurrent electrophoretic separation of said biopolymer fragments within each of said plurality of biopolymer fragment samples, said separation occurring along migration lanes in said electrophoresis module;

(b) means for substantially simultaneously stimulating light emissions from said biopolymer fragments;

(c) a transmission imaging spectrograph for producing grouped signals from substantially simultaneous resolution of said light emissions from said biopolymer fragments into spatial bins representative of said migration lanes and spectral bins representative of said light emissions from said biopolymer fragments, said spatial and spectral bins being adjustable; and

(d) a processor for comparing said grouped signals with a plurality of event prototypes and for selecting the one or more event prototypes closest to said grouped signals.

14. The apparatus of claim 13 wherein said biopolymer fragments are labeled with one or more fluorescent labels, and wherein said means for stimulating light emissions further comprises a laser capable of causing fluorescence of said fluorescent labels.

15. The apparatus of claim 13 wherein said spatial and spectral bins are adjustable.

16. The apparatus of claim 15 wherein said spectral bins are adjusted according to the light emission maxima of fluorescent dyes with distinguishable emission maxima.

17. The apparatus of claim 13 wherein said biopolymer fragments have distinguishable dye labels from which said light emissions are stimulated, and wherein said processor further comprises:

(a) a memory for storing said grouped signals and said plurality of event prototypes;

(b) means for outputting identities of selected event prototypes; and

wherein said processor further provides for (i) filtering and converting said stored grouped signals into a plurality of further signals, said further signals representative of the dye label concentrations, for (ii) comparing the time behavior of said further signals with the time behaviors of said plurality of event prototypes, and for (iii) selecting one or more event prototypes closest to said further signals.

18. The apparatus of claim 17 wherein said biopolymer is DNA, and wherein said memory has stored therein said plurality of event prototypes generated from analyses of known DNA.

19. The apparatus of claim 18 wherein said event prototypes further comprise signals representative of pairs or triples of sequential DNA nucleotides, whereby said selected event prototypes represent the sequence of said DNA fragments.

20. The apparatus of claim 17 wherein said comparing further comprises said processor providing for a distance between said time behaviors of said further signals and said time behaviors of said event prototypes, and wherein said selecting further comprises selecting those one or more event prototypes with substantially smallest distances.

21. The apparatus of claim 20 wherein said distances comprise the weighted sum of the squares of the differences between values of said further signals and values of said event prototypes.

22. The apparatus of claim 13 wherein said transmission imaging spectrograph further comprises:

(a) an optic assembly comprising a transmission dispersion element selected from the group consisting of a transmission grating-prism and a transmission diffraction grating, said optic assembly for substantially simultaneous spatially imaging and spectrally dispersing said light emissions onto a detector array, said spatial imaging being along a first axis, and said spectral dispersion being into rays of differing wavelength and along a second axis; and

(b) said detector array for producing output signals representative of said spatially imaged and spectrally dispersed light emissions.

23. The apparatus of claim 22 wherein said detector array comprises a two-dimensional CCD detector array having detector elements, and wherein said output signals representative of said spatial and spectral bins comprise signals summed from an adjustable one or more adjacent detector elements.

24. The apparatus of claim 22 wherein said optic assembly further comprises:

(a) a collection lens for receiving and collimating said light emissions before spectral dispersion by said transmission dispersion element; and

(b) a focusing lens for focusing said light emission onto said detector array after spectral dispersion by said transmission dispersion element.

25. The apparatus of claim 24 wherein said collection lens and said focusing lens demagnify said light emissions onto said detector array.

26. The apparatus of claim 24 further comprising a spectral filter element for filtering extraneous light from said light emissions.

27. The apparatus of claim 22 wherein said optic assembly further comprises binary optics for spatially focusing and spectrally dispersing said light emissions onto said detector array.

28. The apparatus of claim 13 wherein said migration lanes contain a separation medium configured with a plurality of sample loading wells, and wherein said electrophoresis module further comprises a gap for affording access to said wells for loading of said wells with said biopolymer fragment samples.

29. The apparatus of claim 28 further comprising a solid phase loading system comprising a comb having a plurality of teeth, each tooth within said plurality of teeth being spaced and sized to fit into a different one of said plurality of sample loading wells.

30. The apparatus of claim 28 further comprising a solid phase loading system comprising a plurality of magnetic beads, said beads having means for coupling of said biopolymer fragment samples.

31. The apparatus of claim 13 further comprising a temperature control subunit for maintaining said electrophoresis module at a preselected and substantially uniform temperature.

32. The apparatus of claim 13 wherein said electrophoresis module comprises:

(a) a first substantially flat plate; and

(b) a second substantially flat plate, said first plate being positioned away from said second plate so as to define a rectangular sheet for containing electrophoretic separation medium containing said migration lanes.

33. The apparatus of claim 32 wherein said first and second plates are separated by not less than approximately 25 μ

m.

34. The apparatus of claim 13 wherein said electrophoresis module comprises:

(a) a first substantially flat plate; and

(b) a second substantially flat plate having a plurality of non-intersecting grooves on one side, said first plate being positioned in contact with said second plate so that said grooves form with said first plate a plurality of separated channels, each channel suitable for holding an electrophoretic medium and for forming one of said migration lanes.

35. The apparatus of claim 34 wherein said grooves in said second plate are not less than approximately 10 μ

m in cross-section.

36. The apparatus of claim 34 wherein said grooves in said second plate are straight and spaced to be substantially parallel to each other.

37. The apparatus of claim 34 wherein said grooves in said second plate are spaced to converge toward one end of the second plate.

38. The apparatus of claim 34 wherein said second plate is glass and said grooves are formed by etching said glass.

39. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium, said means for loading comprising

(i) a plurality of wells in the electrophoretic separation medium from which the biopolymer fragment samples migrate for separation, each well containing a buffer medium, and

(ii) a solid phase loading comb having a plurality of teeth, each tooth being spaced and sized to fit into one of the plurality of wells, the teeth having means for the adhesion of the biopolymer fragment samples, the adhesion being such that the fragment samples are released upon insertion into the buffer medium in the wells, and

(iii) notches formed on a plate for guiding the solid phase loading comb into the sample wells, the notches being sized to match the teeth of the comb;

(b) means for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium;

(c) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples; and

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components and generation of output signals representative thereof.

40. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium, said means for loading comprising

(i) a plurality of wells in the electrophoretic separation medium from which the biopolymer fragment samples migrate for separation, each well containing a buffer medium, and

(ii) a solid phase loading comb having a plurality of teeth, each tooth being spaced and sized to fit into one of the plurality of wells, the teeth having means for the adhesion of the biopolymer fragment samples, the adhesion being such that the fragment samples are released upon insertion into the buffer medium in the wells, and

(iii) notches formed on a plate for guiding the solid phase loading comb into the sample wells, the notches being sized to match the teeth of the comb;

(b) means for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium;

(c) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples; and

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components and generation of output signals representative thereof;

wherein the means for the adhesion of the samples is a plurality of DNA sequencing templates bound to the teeth of the comb, said DNA sequencing templates being suitable for adhesion of DNA sequencing reaction fragment samples.

41. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium, said means for loading comprising

(i) a plurality of wells in the electrophoretic separation medium from which the biopolymer fragment samples migrate for separation, each well containing a buffer medium, and

(ii) a solid phase loading system comprising a plurality of magnetic beads, said beads being placed into the plurality of wells, the beads having means for adhesion of the biopolymer fragment samples, the adhesion being such that the fragment samples are released upon insertion into the buffer medium in the wells;

(b) means for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium;

(c) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples; and

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components and generation of output signals representative thereof.

42. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium, said means for loading further comprising means for generating said biopolymer fragment samples;

(b) means for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium;

(c) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples; and

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components and generation of output signals representative thereof.

43. The apparatus of claim 100 wherein the means for generating the plurality of biopolymer fragment samples for separation comprises:

(a) an array of micro-reactors in which biopolymer fragment samples are generated from biopolymer samples, each micro-reactor having a minute inlet for loading a biopolymer sample;

(b) a plurality of capillary inlet passages, each micro-reactor having an inlet passage, through which reagents needed for fragment generation are loaded;

(c) a plurality of capillary outlet passages, each micro-reactor having an outlet passage, through which biopolymer fragment samples are ejected into the electrophoresis module for separation; and

(d) a plurality of capillary controllers, one controller in each capillary inlet and outlet passage, for controlling fluid flow in the capillaries.

44. The apparatus of claim 43 wherein the capillary controller for controlling fluid flow in a capillary comprises:

(a) an electrical micro-heating element in thermal contact with the capillary; and

(b) electrical leads to the heating elements for energizing the heating elements, whereby current in the leads to a micro-heating element causes fluid evaporation in the contacted capillary and formation of a vapor bubble which blocks fluid flow in the capillary.

45. The apparatus of claim 44 wherein the micro-heating elements comprise:

(a) a layer of a resistive material deposited adjacent to the contacted capillary; and

(b) a layer of protective material deposited over the layer of resistive material for separating the resistive layer from the capillary contents.

46. An integrated apparatus for concurrent separation and analysis of a plurality of DNA fragment samples, each said sample comprising a plurality of DNA fragments generated in DNA sequencing reactions or in DNA fingerprinting reactions, the apparatus comprising:

(a) an electrophoresis module for the substantially concurrent electrophoretic separation of said DNA fragments within each of said plurality of DNA fragment samples, said separation occurring along migration lanes in said electrophoresis module;

(b) means for substantially simultaneously stimulating light emissions from said DNA fragments;

(c) a transmission imaging spectrograph for producing grouped signals from substantially simultaneous resolution of said light emissions from said DNA fragments into spatial bins representative of said migration lanes and spectral bins representative of said light emissions from said DNA fragments, said spatial and spectral bins being adjustable; and

(d) a processor for comparing said grouped signals with a plurality of event prototypes and for selecting the one or more event prototypes closest to said grouped signals.

47. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium;

(b) means for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium;

(c) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples;

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components; and

(e) means for the analysis of the detected light emissions to give information on identity of the biopolymer samples and generation of output signals representative thereof;

wherein the biopolymer samples are DNA samples, the biopolymer fragment samples are DNA sequencing reaction fragments labeled with dyes, each dye having distinctive spectral properties, and wherein said means for the analysis further comprises;

(i) memory means for storing the output signals of the means for resolution and detection and for storing a set of prototype signals;

(ii) means for cumulating the stored output signals into spectral samples, each such spectral sample representative of the distinctive spectral characteristics of the dye labels of one biopolymer fragment sample;

(iii) means for comparing the time behavior of the spectral samples with the time behaviors of the set of prototype signals and for selecting prototypes from the set that most closely match the spectral samples; and

(iv) means for outputting identities of the selected prototypes as the DNA sequences of the DNA samples.

48. The apparatus of claim 47 wherein the set of prototype signals comprises the output from the analysis of well known DNA sequences.

49. The apparatus of claim 47 wherein the selected prototypes represent pairs or triples of sequential DNA bases.

50. The apparatus of claim 47 wherein the means for selecting comprises comparing a distance metric between the time behaviors of the spectral samples and the time behaviors of the prototypes and selecting as representative that prototype with the closest distance.

51. The apparatus of claim 50 wherein the distance metric is the sum of the squares of the differences in signal values between the spectral samples and the prototype signals.

52. The apparatus of claim 51 further comprising:

(a) means for trimming from the output DNA sequences of the DNA samples stored in memory known DNA sequences in the DNA sample;

(b) means for proofreading in a Monte Carlo manner the trimmed DNA sequences stored in memory, the means for proofreading comprising means for repetitively making at a random point in the trimmed output a random sequence alteration and evaluating sequence improvement until no further substantial sequence improvement occurs; and

(c) means for storing and outputting the improved sequences.

53. The apparatus of claim 52 wherein sequence improvement is evaluated by evaluating a probabilistic Boltzman condition on the difference in two distance metrics, one distance metric being between the original DNA sequence and the spectral samples, the other distance metric being between alternative DNA sequences and the spectral samples. templates being suitable for adhesion of DNA sequencing reaction fragment samples.

54. An integrated apparatus for concurrent separation and analysis of a plurality of biopolymer fragment samples, each sample comprising a plurality of fragments obtained from one or more biopolymers, the apparatus comprising:

(a) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium;

(b) means for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium;

(c) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples;

(d) means for substantially simultaneous resolution of said light emissions into spatial and spectral components; and

(e) means for the analysis of the detected light emissions to give information on identity of the biopolymer samples and generation of output signals representative thereof;

wherein the biopolymer samples are DNA samples, the biopolymer fragment samples are DNA sequencing reaction fragments labeled with dyes, each dye having distinctive spectral properties, and wherein said means for the analysis further comprises;

(i) memory means for storing the output signals of the means for resolution and detection and for storing a set of prototype signals;

(ii) means for cumulating the stored output signals into a time series of spectral samples, each such spectral sample representative of the distinctive spectral characteristics of the dye labels of one biopolymer fragment sample;

(iii) means for comparing at a plurality of successive observation times the time behavior of the time series of spectral samples with the time behaviors of the set of prototype signals and for selecting a prototype from the set that most closely matches the spectral samples; and

(iv) means for outputting the identity of the prototype that is the closest match.