Method for identifying polymorphisms

• US 6,825,009 B2
• Filed: 03/22/2002
• Issued: 11/30/2004
• Est. Priority Date: 10/01/1998
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Variagenics, Inc. (Arca Biopharma, Inc.)
• Inventors: Verdine, Gregory L., Kawate, Tomohiko, Stanton, Vince P. Jr., Dykes, Colin W, Olson, Jeffrey, Wolfe, Jia Liu
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/105,101
• Publication Number:
  US 20030134290A1
• Time in Patent Office: 984 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.321, 536/26.6
• US Class Current: 435/91.1
• 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 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• 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 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

The present invention relates to methods for the detection of polymorphism in polynucleotides by using hybridization of fragments of segments of a polynucleotide suspected of containing a polymorphism with an oligonucleotide having a sequence complementary to a fragment identifying the polymorphism and subsequent detection of incorporated labels in the oligonucleotide-fragment duplex.

Claims

1. A method for detecting polymorphism in a polynucleotide, comprising:

providing a polynucleotide suspected of containing a polymorphism;

amplifying a segment of the polynucleotide encompassing the suspected polymorphism wherein amplification comprises replacing one or more natural nucleotide(s), one of which is a nucleotide involved in the suspected polymorphism, at substantially each point of occurrence in the segment with a modified nucleotide or, if more than one natural nucleotide is replaced, with different modified nucleotides to form an amplified modified segment;

cleaving the amplified modified segment into fragments by contacting it with a reagent or reagents that cleave(s) the segment at substantially each point of occurrence of the modified nucleotide(s);

hybridizing the fragments to an oligonucleotide; and

,

analyzing the hybridized fragments for an incorporated detectable label identifying the suspected polymorphism.

2. The method of claim 1, wherein the detectable label is incorporated during amplification.

3. The method of claim 2, wherein incorporating the detectable label during amplification comprises using a detectably labeled primer.

4. The method of claim 3, wherein the detectably labeled primer comprises a radioactive primer or a primer containing a fluorophore.

5. The method of claim 1, wherein incorporating the detectable label during amplification comprises using a detectably labeled, modified nucleotide.

6. The method of claim 5, wherein the detectably labeled, modified nucleotide comprises a radioactive modified nucleotide or a modified nucleotide containing a fluorophore.

7. The method of claim 5, wherein the detectably labeled, modified nucleotide is a detectably labeled, modified ribonucleotide.

8. The method of claim 7, wherein the detectably labeled, modified ribonucleotide comprises a radioactive modified ribonucleotide or a modified ribonucleotide containing a fluorophore.

9. The method of claim 1, wherein incorporating the detectable label during amplification comprises replacing a natural nucleotide, that is different than the natural nucleotide(s) being replaced with a modified nucleotide(s), at one or more point(s) of occurrence in the segment with a detectably labeled nucleotide.

10. The method of claim 9, wherein the detectably labeled nucleotide comprises a radioactive nucleotide or a nucleotide containing a fluorophore.

11. The method of claim 9, wherein the detectably labeled nucleotide comprises a delectably labeled ribonucleotide.

12. The method of claim 11, wherein the delectably labeled ribonucleotide comprises a radioactive ribonucleotide or a ribonucleotide containing a fluorophore.

13. The method of claim 1, wherein the detectable label is incorporated during cleavage.

14. The method of claim 13, wherein incorporating the detectable label during cleavage comprises using detectably labeled tris(carboxyethyl)phosphine (TCEP).

15. The method of claim 14, wherein using delectably labeled TCEP comprises using radioactive TCEP or TCEP containing a fluorophore.

16. The method of claim 13, wherein incorporating the detectable label during cleavage comprises using a delectably labeled secondary amine.

17. The method of claim 16, wherein using a detectably labeled secondary amine comprises using a radioactive secondary amine or a secondary amine containing a fluorophore.

18. The method of claim 1, wherein the detectable label is incorporated during hybridization.

19. The method of claim 18, wherein incorporating the detectable label during hybridization comprises hybridizing a second, detectably labeled oligonucleotide to the fragments hybridized to the oligonucleotide.

20. The method of claim 19, wherein the second, detectably labeled oligonucleotide comprises a radioactive oligonucleotide or an oligonucleotide containing a fluorophore.

21. The method of claim 1, wherein the detectable label is incorporated after cleavage or after hybridization, the method comprising:

cleaving using a reagent comprising TCEP or a secondary amine; and

,

substituting the TCEP or secondary amine with a radioactive molecule or a fluorophore after cleavage or after hybridization.

22. The method of claim 1, wherein the polymorphism is selected from the group consisting of a single nucleotide polymorphism (SNP), a deletion or an insertion.

23. The method of claim 1, wherein amplifying the segment comprises a polymerase chain reaction (PCR).

24. The method of claim 1, wherein amplifying the segment comprises replacing one natural nucleotide that is involved in the suspected polymorphism at each point of occurrence in the segment with a modified nucleotide to form a modified segment.

25. The method of claim 24, wherein the modified nucleotide comprises a labeled, modified nucleotide.

26. The method of claim 25, wherein the labeled modified nucleotide comprises a radioactive modified nucleotide or a modified nucleotide containing a fluorophore.

27. The method of claim 24, wherein the modified nucleotide comprises a modified ribonucleotide.

28. The method of claim 24, wherein the modified nucleotide comprises a labeled, modified ribonucleotide.

29. The method of claim 28, wherein the labeled, modified ribonucleotide comprises a radioactive ribonucleotide or a ribonucleotide containing a fluorophore.

30. The method of claim 1, wherein hybridizing the fragments to an oligonucleotide comprises using an oligonucleotide that is immobilized on a solid support.

31. The method of claim 1, wherein the incorporated detectable label comprises fluorescence resonance energy transfer (FRET).