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

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.

What is claimed is:  
     
         1 . An integrated apparatus for concurrent preparation 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 preparing from an input sample of a plurality of biopolymers a plurality of biopolymer fragment samples for subsequent analysis;    (b) means for loading the plurality of biopolymer fragment samples into an electrophoretic separation medium;    (c) means for substantially concurrent electrophoretic separation of each of the plurality of biopolymer fragment samples loaded into the electrophoretic separation medium;    (d) means for substantially simultaneously stimulating light emissions from fragments in a plurality of biopolymer fragment samples; and    (e) means for substantially simultaneous resolution of said light emissions into spatial and spectral components and generation of output signals representative thereof.    
     
     
         2 . The apparatus of  claim 1  further comprising means for the analysis of the detected light emission to give information on identity of the biopolymer samples.  
     
     
         3 . The apparatus of  claim 1  wherein the means for loading the plurality of biopolymer fragment samples comprises: 
 (a) a plurality of wells in the electrophoretic separation medium from which the biopolymer fragment samples migrate for separation, each well containing a buffer medium;  
 (b) 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.  
 
     
     
         4 . The apparatus of  claim 3  wherein the biopolymer fragment samples are DNA sequencing reaction fragment samples, the means for the adhesion of the samples are a plurality of sequencing templates bound to the teeth of the comb, the release upon insertion into the wells occurring with denaturation of the bound fragments from the templates.  
     
     
         5 . The apparatus of  claim 3  in which the solid phase loading comb is guided into the sample wells by notches formed on a plate, the notches being sized to match the teeth of the comb.  
     
     
         6 . The apparatus of  claim 1  wherein the means for loading the plurality of biopolymer fragment samples comprises: 
 (a) 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  
 (b) a solid phase loading system comprising a plurality of magnetic beads, such 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.  
 
     
     
         7 . The apparatus of  claim 1  wherein the means for substantially concurrent electrophoretic separation is a electrophoretic module comprising: 
 (a) a substantially flat bottom plate; and  
 (b) a substantially flat top plate, the top plate being positioned above the bottom plate for forming a narrow cavity to hold the electrophoretic separation medium.  
 
     
     
         8 . The apparatus of  claim 7  wherein the top and bottom plates are separated by approximately 25 μm to approximately 250 μm.  
     
     
         9 . The apparatus of  claim 7  further comprising means for thermal control for maintaining a selected uniform temperature in the bottom plate.  
     
     
         10 . The apparatus of  claim 9  wherein the means for maintaining thermal control in the electrophoresis module further comprises: 
 (a) a heat sink for exchanging heat with the surroundings; and  
 (b) 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.  
 
     
     
         11 . The apparatus of  claim 10  wherein the thermal transfer devices are Peltier thermo-electric devices.  
     
     
         12 . The apparatus of  claim 7  wherein electrophoretic migration lanes are formed between the top and bottom plates.  
     
     
         13 . The apparatus of  claim 1  wherein the means for substantially concurrent electrophoretic separation is an electrophoresis module comprising: 
 (a) a substantially flat bottom plate; and  
 (b) 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.  
 
     
     
         14 . The apparatus of  claim 13  wherein the grooves in the top plate are from approximately 25 μm to approximately 250 μm in cross-section.  
     
     
         15 . The apparatus of  claim 13  wherein the grooves in the top plate are straight and spaced to be parallel.  
     
     
         16 . The apparatus of  claim 13  wherein the grooves in the top late are spaced to converge toward one end of the plate.  
     
     
         17 . The apparatus of  claim 13  further comprising: 
 (a) 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  
 (b) 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.  
 
     
     
         18 . The apparatus of  claim 13  in which either the top plate or the bottom plate is a thermal conductor coated with an electrical insulator, and the grooves are formed on the insulator.  
     
     
         19 . The apparatus of  claim 13  in which the separation medium comprises small posts fabricated directly in the migration lanes.  
     
     
         20 . The apparatus of  claim 1  in which the separation medium comprises small spheres of an inert material such as polystyrene.  
     
     
         21 . The apparatus of  claim 1  wherein the means for substantially simultaneous resolution of each of a plurality of light emissions is a transmission imaging spectrograph comprising: 
 (a) 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  
 (b) 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.  
 
     
     
         22 . The apparatus of  claim 21  wherein the detector array is a CCD array producing electronic output signals.  
     
     
         23 . The apparatus of  claim 21  wherein the optic assembly further comprises: 
 (a) a collection lens positioned to initially receive and collimate the plurality of light emissions;  
 (b) a transmission dispersion element for spectrally dispersing the collimated signals along the first axis; and  
 (c) a focusing lens for spatially focusing the signals along the second axis onto the detector array.  
 
     
     
         24 . The apparatus of  claim 23  further comprising a spectral filter element positioned between the collection lens and transmission dispersion element for filtering from the light emissions extraneous light.  
     
     
         25 . The apparatus of  claim 23  in which the transmission dispersion element is a transmission diffraction grating, or a transmission grating-prism.  
     
     
         26 . The apparatus of  claim 21  wherein the optic assembly further comprises binary optics for spectrally dispersing the light emissions along the first axis and for focusing the light emissions along the second axis onto the detector array.  
     
     
         27 . The apparatus of  claim 1  wherein the biopolymer fragment samples are labeled with spectrally distinctive dyes and further comprising a laser illuminating the separated biopolymer fragment samples to stimulate light emission from the dye labels.  
     
     
         28 . The apparatus of  claim 1  wherein the means for loading 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.  
 
     
     
         29 . The apparatus of  claim 28  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.  
 
     
     
         30 . The apparatus of  claim 29  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.  
 
     
     
         31 . The apparatus of  claim 1  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, said apparatus further comprising: 
 (a) memory means for storing the output signals of the means for resolution and detection and for storing a set of prototype signals;  
 (b) 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,  
 (c) 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  
 (d) means for outputting identities of the selected prototypes as the DNA sequences of the DNA samples.  
 
     
     
         32 . The apparatus of  claim 31  wherein the set of prototype signals comprises the output from the analysis of well known DNA sequences.  
     
     
         33 . The apparatus of  claim 31  wherein the selected prototypes represent pairs or triples of sequential DNA bases.  
     
     
         34 . The apparatus of  claim 31  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.  
     
     
         35 . The apparatus of  claim 34  wherein the distance metric is the sum of the squares of the differences in signal values between the spectral samples and the prototype signals.  
     
     
         36 . The apparatus of  claim 35  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.  
 
     
     
         37 . The apparatus of  claim 36  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.  
     
     
         38 . The apparatus of  claim 1  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, said apparatus further comprising: 
 (a) memory means for storing the output signals of the means for resolution and detection and for storing a set of prototype signals;  
 (b) 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;  
 (c) 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  
 (d) means for outputting the identity of the prototype that is the closest match.  
 
     
     
         39 . A method for generating DNA sequence reaction fragments in one reaction chamber without an intermediate separation step the method comprising the sequential steps of: 
 (a) performing the polymerase chain reaction amplification step with dUTP rich PCR primers;    (b) fragmenting the dUTP primers with Uracil DNA Glycosylase into fragments ineffective as DNA polymerase primers; and    (c) performing the Sanger sequencing reactions.    
     
     
         40 . The method of  claim 39  wherein the dUTP rich PCR primers have dUTP residues spaced no more than approximately six bases apart.  
     
     
         41 . The method of  claim 39  performed in an array of micro-reactors for ejection onto a biopolymer separation apparatus.  
     
     
         42 . A method for determining the DNA sequences of a plurality of DNA samples, the method using spectral signals obtained by spectrographic observation of electrophoretically separated labeled DNA fragments, the fragments being produced by the Sanger sequencing reactions and being labeled with dyes having distinctive spectral properties, the method comprising the sequential steps of: 
 (a) cumulating the spectrographic signals into a plurality of spatial samples, each spatial sample being representative of fragments of one DNA sample, and for each spatial sample, cumulating the spectrographic output signals into spectral samples, each spectral sample being representative of the distinctive spectral characteristics of one of the dye labels;    (b) comparing the time behavior of the spectral samples with a set of prototype signal time behaviors and selecting prototypes from the set that most closely match the spectral samples; and    (c) outputting the identities of the selected prototypes.    
     
     
         43 . The method of  claim 42  wherein the time behaviors of the set of prototype signals comprises the grouped output from the analysis of well known DNA sequences.  
     
     
         44 . The method of  claim 43  wherein the selected prototypes are pairs of sequential DNA fragments.  
     
     
         45 . The method of  claim 42  wherein the step of 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.  
     
     
         46 . The method of  claim 45  wherein the distance metric is the sum of the squares of the differences in signal values between the spectral samples and the prototypes.  
     
     
         47 . The method of  claim 45  wherein the distance metric is the product of (x), the sum of the squares of the differences in signal values between the spectral samples and the prototypes, and (y) a metric distance representing the difference between the prototypically expected time between observation times of closest match, and the actual time between observation times of closest match.  
     
     
         48 . The method of  claim 47  in which the metric distance (y) is a function of the square of the difference between the prototypically expected time between observation times of closest match and the actual time between observation times of closest match.  
     
     
         49 . The method of  claim 41  further comprising the steps of: 
 (a) trimming from the output identities of the DNA samples known DNA sequences in the DNA sample; and  
 (b) proofreading in a Monte Carlo manner the trimmed DNA sequences stored in memory, the step of 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) storing and outputting the improved sequence.  
 
     
     
         50 . The method of  claim 49  wherein evaluating sequence improvement is done 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.  
     
     
         51 . A method for determining the DNA sequences of a plurality of DNA samples, the method using spectral signals obtained by spectrographic observation of electrophoretically separated labeled DNA fragments, the fragments being produced by the Sanger sequencing reactions and being labeled with dyes having distinctive spectral properties, the method comprising the sequential steps of: 
 (a) storing the output signals of the means for resolution and detection and for storing a set of prototype signals;    (b) 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;    (c) 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 selecting a prototype from the set that most closely matches the spectral samples; and    (e) outputting the identity of the prototype that is the closest match.    
     
     
         52 . 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 bidirectional 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.  
   
     
     
         53 . The apparatus of  claim 52  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.