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

• US 6,236,945 B1
• Filed: 04/26/1999
• Issued: 05/22/2001
• 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): Sisson, Bradley L.
• Application Number:
  US09/300,703
• Time in Patent Office: 757 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/194, 435/287.2, 436/94, 436/800, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 702/20
• US Class Current: 702/20
• 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. A method for determining the DNA sequences of a plurality of DNA samples, the method using spectral signals obtained by spectrographic detection of electrophoretically separated labeled DNA fragments in a plurality of migration lanes, the fragments in each of said samples being produced from one or more DNA templates primed with one or more sequencing primers and being labeled with dyes having distinctive spectral properties, the method comprising the sequential steps of:

(1) binning spectrographic signal intensities acquired at each of a plurality of observation times from each of said plurality of migration lanes into a spatial signal set to obtain a plurality of spatial signal sets for each said observation time and into spectral signal sets where each spatial signal set represents the distinctive spectral properties of one of a plurality of said dyes, wherein each spatial signal set and each spectral signal set contain signal intensities from one migration lane, wherein each migration lane contains the fragments from one DNA sample, said plurality of dyes being used to label fragments in said sample;

(2) repeating step (1) for spectrographic signal intensities acquired at each observation time to obtain time behaviors of said spectral signals;

(3) comparing for each spatial signal set the time behaviors of said spectral signals in each of said plurality of spatial signal sets with time behaviors of a set of prototype signals corresponding to a set of prototypes;

(4) selecting prototypes from said set of prototype signals that most closely match said spectral signals; and

(5) outputting the selected prototypes, thereby determining the sequences of said plurality of DNA samples;

wherein said spectrographic signal intensities are input from a transmission imaging spectrograph which detected signal intensities measured simultaneously for said plurality of migration lanes and for said plurality of spectral values at each of said plurality of observation times; and

wherein each of said migration lanes contains one or more sets of said labeled DNA fragments, said labeled DNA fragments in each said set being characterized by being produced from a certain DNA template primed with a certain sequencing primer and having a certain nucleotide at the 3′

end, wherein each set of said labeled DNA fragments in each sample is labeled with one or more dyes distinguishable by distinctive spectral properties from the dyes labeling the other sets of fragments in said sample.

2. The method of claim 1, wherein the time behaviors of the set of prototype signals are obtained from the analysis of known DNA sequences.

3. The method of claim 2 wherein the selected prototypes are pairs of sequential DNA fragments.

4. The method of claim 1 wherein the step of selecting comprises determining a distance between the time behaviors of the spectral signals and the time behaviors of the prototypes and selecting as representative that prototype providing the smallest distance.

5. The method of claim 4 wherein the distance is the sum of the squares of the differences in signal values between the spectral signals and the prototype signals.

6. The method of claim 4 wherein the distance is the product of (x), the sum of the squares of the differences in signal values between the spectral signals and the prototype signals, and (y), a distance representing the difference between the expected time between observation times of closest match, and the actual time between observation times of closest match.

7. The method of claim 6 in which the distance (y) is a function of the square of the difference between the expected time between observation times of closest match and the actual time between observation times of closest match.

8. The method of claim 7 further comprising the steps of:

(1) trimming from each of the output sequences of the DNA samples a portion of said sequence said portion being known to be present in the DNA sequencing template;

(2) proofreading each of the trimmed DNA sequences, the step of proofreading comprising repetitively making at a random point in the trimmed DNA sequence a random sequence alteration and evaluating sequence improvement until no further substantial sequence improvement occurs; and

(3) outputting each improved sequence.

9. The method of claim 8 wherein evaluating sequence improvement is done by comparing an energy function of a distance between the signals of original DNA sequence and the spectral signals with an energy function of said distance between the signals of said trimmed DNA sequence having a random sequence alteration and the spectral signals.

10. The method of claim 1, wherein each one of said plurality of migration lanes contains the fragments produced from one DNA sequencing template.

11. The method of claim 1 or 10, wherein said fragments are generated by a Sanger sequencing reaction.

12. The method of claim 11, wherein said Sanger sequencing reaction uses more than one species of dideoxynucleotide, each species of dideoxynucleotide being labeled with a different dye having a distinctive spectral property.

13. The method of claim 1 or 10, wherein said fragments are generated by PCR amplification and subsequent exonuclease digestion.

14. The method of claim 13, wherein said fragments are generated using more than one primer, each primer being labeled with a different dye having a distinctive spectral property.

15. The method of claim 13, wherein said fragments are generated using more than one blocking base analog, each blocking base analog being labeled with a different dye having a distinct emission spectrum.