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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.