Method, apparatus and kits for sequencing of nucleic acids using multiple dyes

• US 20030003498A1
• Filed: 08/12/2002
• Published: 01/02/2003
• Est. Priority Date: 04/18/1996
• Status: Abandoned Application

• Current Assignee: Bayer Healthcare LLC (Bayer AG)
• Original Assignee: Bayer Healthcare LLC (Bayer AG)
• Inventors: Digby, Thomas J., Izmailov, Alexandre
• Application Number:
  US10/217,615
• Publication Number:
  US 20030003498A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation

Abstract

An instrument for sequencing oligonucleotides is loaded with the products of four sequencing reaction mixtures. These products are a combination of A, C, G and T reaction products for several sequencing reactions. The products of the different sequencing reactions are labeled with fluorescent tags which are distinguishable one from the other on the basis of their excitation or emission spectra. After separation of the oligonucleotides by electrophoresis, the order of the detected peaks is used to call the base sequence.

Claims

1. A method for evaluating the nucleic acid sequence of a plurality of samples comprising the steps of

(a) obtaining a first aliquot of each sample;

(b) combining the first aliquot of each sample with a sequencing reaction mixture containing a polymerase enzyme, a primer for hybridizing with the sample, nucleoside feedstocks and a first dideoxynucleoside to form a first plurality of mixtures of product oligonucleotide fragments, one for each sample, wherein the product oligonucleotide fragments formed from each first aliquot are labeled with different fluorescent tags, said different fluorescent tags being distinguishable one from the other on the basis of their excitation or emission spectra;

(c) combining the first plurality of mixtures of oligonucleotide products to form a first combined mixture;

(d) loading the first combined mixture onto a separation matrix at a first loading site;

(e) applying an electric field to cause the oligonucleotide products to migrate within the separation matrix; and

(f) detecting the oligonucleotide products having the different fluorescent tags as they migrate within the separation matrix.

2. The method according to claim 1, further comprising the steps

(i) obtaining a second aliquot of each sample;

(ii) combining the second aliquot of each sample with a sequencing reaction mixture containing a polymerase enzyme, a primer for hybridizing with the sample, nucleoside feedstocks and a second dideoxynucleoside different from the first dideoxynucleoside to form a second plurality of mixtures of product oligonucleotide fragments, one for each sample, wherein the product oligonucleotide fragments formed form each second aliquot are labeled with different fluorescent tags, said different fluorescent tags being distinguishable one from the other on the basis of their excitation or emission spectra;

(iii) combining the second plurality of mixtures of oligonucleotide products to form a second combined mixture;

(iv) loading the second combined mixture onto a separation matrix at a second loading site, distinct from the first loading site;

(v) applying an electric field to cause the oligonucleotide products to migrate within the separation matrix; and

(vi) detecting the oligonucleotide products having the different fluorescent tags as they migrate within the separation matrix.

3. The method according to claim 2, wherein the first and second aliquots are combined with a sequencing reaction concurrently, and wherein the first and second combined mixtures are loaded onto different lanes of the same gel.

4. The method according to claim 3, wherein the same primer and fluorescent tag are combined with the first and second aliquots of each sample.

5. The method according to claim 1, wherein first aliquots from at least three samples are combined to form the first combined mixture.

6. The method according to claim 1, wherein first aliquots from more than four samples are combined to form the first combined mixture.

7. A method for evaluating the sequence of a target nucleic acid polymer in a plurality of samples comprising the steps of

(a) obtaining four aliquots of each sample;

(b) combining the aliquots of each sample with four sequencing reaction mixtures, each sequencing reaction mixture containing a polymerase enzyme, a primer for hybridizing with the target nucleic acid, nucleoside feedstocks and a different dideoxynucleoside to form an A-mixture, a G-mixture, a T-mixture and a C-mixture for each sample containing product oligonucleotide fragments of varying lengths, wherein the product oligonucleotide fragments are labeled with fluorescent tags, and the fluorescent tags used for each sample are distinguishable one from the other on the basis of their excitation or emission spectra;

(c) combining the A-mixtures, the C-mixtures, the T-mixtures and the C-mixtures for each sample to form a combined A-mixture, a combined C-mixture, a combined t-mixture and a combined C-mixture;

(d) loading the combined A-mixture, the combined G-mixture, the combined T-mixture and the combined C-mixture onto a separation matrix at separate loading sites;

(e) applying an electric field to cause the product oligonucleotide fragments to migrate within the separation matrix; and

(f) detecting the product oligonucleotide fragments having the different fluorescent tags as they migrate within the separation matrix.

8. An apparatus for evaluating the sequence of a nucleic acid polymer by separation of a reaction mixture containing oligonucleotide fragments in a separation matrix disposed within the apparatus comprising

(a) means for providing excitation energy to a detection site within the separation matrix;

(b) means for detecting light emitted from fluorescently-labeled oligonucleotide fragments located within the detection zone;

(c) configuration control means, operatively connected to the means for providing excitation energy and the means for detecting to provide combinations of excitation wavelength and detection wavelength specific for a plurality of different fluorescently-labeled oligonucleotide fragments; and

(d) data processing means, operatively connected to the configuration control means and the means for detecting for receiving a signal from the means for detecting and assigning that signal to a data stream based upon the combination of excitation wavelength and detection wavelength set by the configuration control means.

9. A kit for determining the sequence of a selected region of DNA comprising, in packaged combination, at least of first set of a plurality of containers, each container containing a reagent species for the sequencing of the selected region of DNA, wherein the reagent species in each container comprises a reactive portion and a label portion and wherein the label portions of the reagents are different and distinguishable one from the other.

10. The kit according to claim 9, wherein the detectable labels are fluorescent tags, distinguishable on the basis of the excitation or emission spectra thereof.

11. The kit according to claim 10, wherein the reactive portion of each reagent species in the first set of containers is an oligonucleotide primer for use in sequencing the selected region of DNA.

12. The kit according to claim 11, wherein the reactive portions of each reagent species in the first set of containers are the same as one another.

13. The kit according to claim 11, wherein the reactive portion of each reagent species in the first set of containers is a dideoxynucleoside and wherein the reactive portions of each reagent species in the first set of containers are the same as one another.

14. The kit according to claim 13, further comprising a second set of containers, each containing a reagent species for the sequencing of the selected region of DNA, wherein the reagent species in each container of the second set comprises a dideoxynucleoside reactive portion different from the reactive portion of the reagent species in the first set of containers, and a fluorescent label portion and wherein the label portions of the reagents in the second set of containers are different and distinguishable one from the other on the basis of the excitation or emission spectra thereof.

16. The method according to claim 15, wherein the detectable labels are fluorescent tags.

17. The method according to claim 16, wherein fluorescent tags are distinguishable based upon their excitation or emission spectra.

18. The method according to claim 15, wherein four aliquots of sample are combined with four separate sequencing reaction mixtures, each containing a different dideoxynucleoside.

15. A method for evaluating the sequence of a plurality of gene regions within a sample comprising the steps of:

(a) combining at least a first aliquot of the sample with a sequencing reaction mixture containing a polymerase enzyme, a plurality of sequencing primer species, one sequencing primer species for each gene region, nucleoside feedstocks and a first dideoxynucleoside to form a first mixture of product oligonucleotide fragments, wherein each of the sequencing primer species is labeled with a different detectable label, said different detectable labels being distinguishable one from the other by a detection system;

(b) separating the first mixture of product oligonucleotide fragments based upon the size of the fragments;

(c) detecting emissions from the separated oligonucleotide fragments for each different detectable label; and

(d) evaluating the sequence of each gene region based upon the oligonucleotide fragments detected.

19. A kit for evaluating the sequence of a plurality of gene regions within a sample comprising, in packaged combination, at least one container containing a mixture of a plurality of sequencing primers, one for each gene region to be evaluated, wherein the plurality of sequencing primers each comprise a reactive portion which hybridizes with DNA in the sample and a label portion and wherein the label portions of the reagents are different and distinguishable one from the other.

20. The kit according to claim 20, wherein the detectable labels are fluorescent tags, distinguishable one from the other by their emission or excitation spectra.