Method for determining specific nucleotide variations by primer extension in the presence of mixture of labeled nucleotides and terminators

• US 6,013,431 A
• Filed: 12/02/1993
• Issued: 01/11/2000
• Est. Priority Date: 02/16/1990
• Status: Expired due to Term

• Current Assignee: Laboratory Corporation Of America Holdings
• Original Assignee: Molecular Tool, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Soderlund, Hans E., Syvanen, Anne-Christine
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/162,376
• Time in Patent Office: 2,231 Days
• Field of Search: 435/6, 435/91.2, 435/5, 536/24.33
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• 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 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2563/101 radioactivity, e.g. radioactive labels

Abstract

Detection of variable nucleotide(s) is based on primer extension and incorporation of detectable nucleoside triphosphates. By selecting the detection step primers from the region immediately adjacent to the variable nucleotide, this variation can be detected after incorporation of as few as one nucleoside triphosphate. Labelled nucleoside triphosphates matching the variable nucleotide are added and the incorporation of a label into the detection step primer is measured. The selection of the detection step primer is important to the method according to this invention and is dependent on the nucleotide sequence of interest. The detection step primers are preferably selected so as to span the region immediately toward the 3'"'"' end from the variable nucleotide to be detected. The detection step primers can also be complementary to a sequence beginning several nucleotides removed from the variable nucleotide. The only limitation concerning the position of the detection step primers is that the sequence between the 3'"'"' end of the detection step primer and the variable nucleotide to be detected must not contain a nucleotide residue of the same type as the one to be detected. The detection step primers can equally well be chosen to be complementary to either the coding or non-coding strands of the nucleotide sequence of interest.

Claims

1. A method for determining the identity of a specific nucleotide at one or more defined sites in a target nucleic acid polymer, the method comprising the steps of:

(a) providing a detectable amount of a target nucleic acid polymer in a single stranded form,

(b) hybridizing the detectable amount of the nucleic acid polymer with one or more oligonucleotide primers, wherein each primer has a nucleotide sequence that is complementary to a sequence in the target nucleic acid polymer, such that when the primer is hybridized to the target nucleic acid polymer, the 3'"'"' end of the primer binds to a nucleotide flanking the specific nucleotide at the defined site in the target nucleic acid,

(c) exposing the hybridized nucleic acid polymer to a polymerization agent in a mixture containing at least one deoxynucleotide, said deoxynucleotide comprising a detectable label, and one or more chain terminating nucleotide analogues, such that a detectable primer extension product is formed if the labeled deoxynucleotide is complementary to the specific nucleotide at the defined site, and

(d) analyzing the polymerization mixture of step (c) for the presence or absence of the primer extension product containing the labeled deoxynucleotide at the 3'"'"' end thereof, whereby the identity of the specific nucleotide at the defined site is determined.

2. A method according to claim 1 wherein the mixture of step (c) comprises at least two deoxynucleotides, each comprising a different base and at least one of which is labeled.

3. A method according to claim 1 wherein the primer hybridizes to the target nucleic acid immediately adjacent the specific nucleotide being identified.

4. A method according to claim 1 wherein the detectable amount of target nucleic acid polymer is obtained by performing an amplification reaction.

5. A method according to claim 1 wherein the one or more deoxynucleotides are deoxynucleoside triphosphates selected from among dATP, dGTP, dCTP and dTTP.

6. A method according to claim 1 wherein the one or more chain terminating nucleotide analogues are dideoxynucleoside triphosphates selected from among ddATP, ddGTP, ddCTP and ddTTP.

7. A method according to claim 1 wherein the polymerization agent is a DNA polymerase.

8. A method according to claim 1 or 2, wherein the labeled deoxynucleotide comprises a fluorescent or radioactive label.

9. A method according to claim 1 or 2, wherein the target nucleic acid is immobilizable or immobilized to a solid support.

10. A method according to claim 9 wherein the target nucleic acid comprises an attachment moiety.

11. A method according to claim 10 wherein the attachment moiety is biotin.

12. A method according to claim 1 wherein the primer extension product is removed from the target nucleic acid prior to determining incorporation of the labeled deoxynucleotide.

13. A method according to claim 1 or 2, wherein a plurality of specific nucleotides may be detected at different defined sites in the target nucleic acid polymer by hybridizing a plurality of oligonucleotide primers to complementary sequences in the polymer flanking each site and extending the primers with differently labeled deoxynucleotides.

14. A method according to claim 13, wherein the target nucleic acid is immobilizable or immobilized to a solid support.

15. A method according to claim 14, wherein the target nucleic acid comprises an attachment moiety.

16. A method according to claim 15 wherein the attachment moiety is biotin.

17. A method according to claim 2 or 13 wherein each deoxynucleotide comprises a different label, the label for each deoxynucleotide being separately detectable.

18. A method according to claim 13 or 14, wherein the specific nucleotides at different defined sites are detected in one single step.

19. A method according to claim 1 wherein the target nucleic acid polymer is derived from a microorganism and the specific nucleotide is a point mutation known to lead to altered pathogenicity or resistance to therapy in the microorganism.

20. A method according to claim 19, wherein the microorganism is human immunodeficiency virus.

21. A method according to claim 20, wherein the point mutation is at a site in the target nucleic acid polymer selected from among the codons encoding Asp 67, Lys 70 and Thr 215.

22. A method according to claim 1 wherein the specific nucleotide in the target nucleic acid polymer is a point mutation in the genetic material of a human cell known to result in a genetic disease or a cancer.

23. A method according to claim 22 wherein the target nucleic acid polymer is obtained or derived from a mixture of mutated and unmutated human cells.

24. A method according to claim 23 wherein the target nucleic acid polymer is obtained or derived from leukemic cells.

25. A method according to claim 22 or 23, wherein the human cells are lymphocytes.

26. A method according to claim 1 wherein the target nucleic acid is obtained or derived from the cells of a patient and the detection of the specific nucleotide indicates predisposition to a genetic disease in the patient.