Fluorescence-based genotyping

• US 6,855,500 B2
• Filed: 03/26/2002
• Issued: 02/15/2005
• Est. Priority Date: 10/01/1998
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Allerson, Charles R., Stanton, Vincent P. Jr., Wolfe, Jia Liu, Kawate, Tomohiko
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US10/107,748
• Publication Number:
  US 20030165880A1
• Time in Patent Office: 1,057 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

The present invention relates to a method for genotyping a diploid organism to detect single nucleotide polymorphisms (SNPs) using modified nucleotides or nucleotide residues substituted with fluorescent groups.

Claims

1. A method for genotyping a diploid organism, comprising:

providing two alleles of a target gene of a diploid organism suspected to contain a polymorphism;

obtaining a first segment of one allele wherein the segment includes the suspected polymorphic locus;

obtaining a second segment from the other allele wherein the segment also includes the suspected polymorphic locus;

replacing a first natural nucleotide, suspected to be at the polymorphic locus, at greater than 90% of its points of occurrence in the first and the second segment with a first modified nucleotide to give a first and a second modified segment;

replacing a second natural nucleotide, suspected to be at the polymorphic locus, at greater than 90% of its points of occurrence in the first and the second modified segments with a second modified nucleotide to give a first and a second twice-modified segment, wherein;

replacing the natural nucleotides with modified nucleotides comprises amplification using primers that hybridize to each segment or to each modified segment such that the suspected polymorphic locus will be the first occurrence of a modified nucleotide after the 3′

end of the primer in each amplified segment;

covalently bonding a first fluorophore that emits light at a first wavelength to the first modified nucleotide or a residue thereof;

covalently bonding a second fluorophore that emits light at a second wavelength to the second modified nucleotide or a residue thereof;

cleaving the first and second twice-modified segments at greater than 90% of the points of occurrence of each modified nucleotide to give a first and second set of fragments, one fragment of each segment comprising primer and a modified nucleotide or residue thereof;

isolating the two primer-containing fragments;

analyzing the isolated fragments for the emission of the first wavelength of light, the second wavelength of light, or both, wherein;

if only one of the wavelengths is detected, then the same modified nucleotide was incorporated at the suspected polymorphic locus in both the first and the second allelic segments indicating that the alleles of the target gene are homozygous, whereas if both wavelengths are detected, then a different modified nucleotide was incorporated at the suspected polymorphic locus in the first and the second allelic segments indicating that the alleles of the target gene are heterozygous.

2. The method of claim 1, wherein isolating the primer-containing fragments comprises immobilizing the primer.

3. The method of claim 2, wherein immobilizing the primer comprises hybridizing the primer to a complementary oligonucleotide that is bound to a solid support.

4. The method of claim 3, wherein the primer is immobilized before amplification.

5. The method of claim 3, wherein the primer is immobilized after amplification but before cleavage.

6. The method of claim 3, wherein the primer is immobilized after cleavage.

7. The method of claim 1, wherein isolating the primer-containing fragments comprises high performance liquid chromatography (HPLC).

8. The method of claim 1, wherein isolating the primer-containing fragments comprises electrophoresis.

9. The method of claim 1, wherein the fluorophores are covalently bonded to the first and second modified nucleotides prior to amplification.

10. The method of claim 9, wherein the fluorophores are covalently bonded to a base moiety of the first, the second or both modified nucleotides.

11. The method of claim 10, wherein cleavage comprises a reagent or reagents that cleave(s) at a 3′

end of the modified nucleotide(s) having a fluorophore bonded to the base.

12. The method of claim 1, wherein the first fluorophore is covalently bonded to the first modified nucleotide prior to amplification and the second fluorophore is bonded to a residue of the second modified nucleotide during cleavage.

13. The method of claim 12, wherein bonding the second fluorophore to a residue of the second modified nucleotide during cleavage, comprises:

cleaving the first and second twice modified segments with a reagent comprising a chemical base and a fluorophore-containing phosphine;

wherein,

the residue of the second modified nucleotide forms a covalent bond with the fluorophore-containing phosphine during cleavage.

14. The method of claim 13, wherein the phosphine comprises tris(2-carboxyethyl)phosphine (TCEP) and the chemical base is a secondary amine.

15. The method of claim 14, wherein the TCEP comprises N¹-(5/6-carboxyfluorescein)-1,6-diaminohexane-TCEP.

16. The method of claim 13, wherein the second modified nucleotide comprises 7-nitro-7-deazadeoxyadenine triphosphate, 7-nitro-7-deazadeoxyguanidine triphosphate, 5-hydroxydeoxycytidine triphosphate, 5-hydroxydeoxyuridine triphosphate or 5-aminodeoxyuridine triphosphate.

17. The method of claim 1, wherein the first fluorophore is covalently bonded to the first modified nucleotide after amplification but before cleavage and the second fluorophore is covalently bonded to a residue of the second modified nucleotide during cleavage.

18. The method of claim 1, wherein the first fluorophore is covalently bonded to a residue of the first modified nucleotide after cleavage and the second fluorophore is bonded to a residue of the second modified nucleotide during cleavage.

19. The method of either claim 17 or claim 18, wherein bonding the first fluorophore to the first modified nucleotide comprises:

a functional group covalently bonded to the first modified nucleotide; and

,

a functional group covalently bonded to the fluorophore that reacts with the functional group on the modified nucleotide to form a bridge of covalently bonded atoms between the fluorophore and the modified nucleotide or modified nucleotide residue.

20. The method of either claim 17 or claim 18, wherein bonding the second fluorophore to the second modified nucleotide residue, comprises:

cleaving the modified nucleotide segments with a reagent comprising a base and a fluorophore-containing phosphine;

wherein,

the second modified nucleotide residue forms a covalent bond with the phosphine during cleavage.

21. The method of claim 20, wherein the phosphine comprises tris(2-carboxyethyl)phosphine (TCEP) and the base is a secondary amine.

22. The method of claim 21, wherein the TCEP comprises N¹-(5/6-carboxyfluorescein)-1,6-diaminohexane-TCEP.

23. The method of claim 20, wherein the second modified nucleotide comprises 7-nitro-7-deazadeoxyadenine triphosphate, 7-nitro-7-deazadeoxyguanidine triphosphate, 5-hydroxydeoxycytidine triphosphate, 5-hydroxydeoxyuridine triphosphate or 5-aminodeoxyuridine triphosphate.

24. The method of any one of claim 12, 17 or 18, wherein bonding the second fluorophore to a residue of the second modified nucleotide during cleavage comprises cleaving with a secondary amine covalently bonded to a fluorophore.

25. The method of claim 1, wherein the fluorophores are bonded to at least 1% of each of the two modified nucleotide or nucleotide residues.

26. The method of claim 1, wherein the fluorophores are bonded to at least 5% of each of the two modified nucleotide or nucleotide residues.