Method for determining specific nucleotide variations

• US 7,026,117 B2
• Filed: 02/26/1999
• Issued: 04/11/2006
• Est. Priority Date: 02/16/1990
• Status: Expired due to Fees

• Current Assignee: Laboratory Corporation Of America Holdings
• Original Assignee: Orchid Biosciences Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Söderlund, Hans E., Syvanen, Anne-Christine
• Primary Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US09/258,216
• Publication Number:
  US 20030082531A1
• Time in Patent Office: 2,601 Days
• Field of Search: 435/5, 435/6, 435/91.2, 435/91.1, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 435/6.11
• 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 detecting the presence of one or more specific nucleotides at a predetermined target position in a target nucleic acid, the method comprising the steps of:

(a) providing an analyzable amount of the target nucleic acid in a single stranded form;

(b) hybridizing the target nucleic acid with a detection primer to form a target-nucleic-acid/detection-primer hybrid, the detection primer comprising a detection-primer nucleotide sequence having a primer-extension-initiation 3′

-end nucleotide which constitutes a 3′

end of the detection primer, the detection-primer nucleotide sequence being complementary to a primer-hybridizing nucleotide sequence of the target nucleic acid with a nucleotide in the target nucleic acid complementary to the primer-extension-initiation 3′

-end nucleotide of the detection-primer nucleotide sequence defining a primer-end complement nucleotide, the primer-hybridizing nucleotide sequence of the target nucleic acid extending towards the 3′

end of the target nucleic acid from the primer-end complement nucleotide, the primer-end complement nucleotide being located in the target nucleic acid at a position 3′

-ward of the predetermined target position, the position of the primer-end complement nucleotide being subject to a constraint that no nucleotide of the same type as the one or more specific nucleotides to be detected be located in the target nucleic acid in any position between the position of the primer-end complement nucleotide and the predetermined target position;

(c) forming an extension-reaction mixture by exposing the target-nucleic-acid/detection-primer hybrid to an admixture of a polymerization agent and a plurality of nucleoside triphosphates, the nucleoside triphosphates of the admixture including at least one deoxynucleotide and at least one chain-terminating nucleotide analogue, at least one deoxynucleotide defining a labeled deoxynucleotide comprising a detectable label or an attachment moiety capable of binding a detectable label, each deoxynucleotide of the admixture of nucleoside triphosphates being complementary to a nucleotide which differs from any nucleotide to which a chain-terminating nucleotide analogue of the admixture is complementary, the plurality of nucleoside triphosphates of the admixture being such that, if a labeled deoxynucleotide is complementary to a specific nucleotide at the predetermined target position, a detectable primer-extension product is formed of the detection primer extended to include an extension portion incorporating the labeled deoxynucleotide; and

(d) analyzing the extension-reaction mixture from step (c) for the presence or absence of a detectable label in association with a labeled deoxynucleotide incorporated in an extension portion of a primer extension product to detect the presence of the corresponding specific nucleotide at the target position in the target nucleic acid.

3. A method according to either of claims 1 or 2, 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.

4. A method according to either of claims 1 or 2, wherein the analyzable amount of target nucleic acid is obtained by performing an amplification reaction on a sample of nucleic acid.

5. A method according to either of claims 1 or 2 wherein the target nucleic acid comprises an attachment moiety.

6. A method according to claim 5 wherein the target nucleic acid is immobilized on a solid matrix during step (b).

7. A method according to claim 5 wherein the target nucleic acid is immobilized on a solid matrix during step (c).

8. A method according to either of claims 1 or 2 in which the primer-end complement nucleotide is located in the target nucleic acid at a position immediately adjacent to the predetermined target position.

9. A method according to either of claims 1 or 2 wherein the detection-primer nucleotide sequence is from 10 to 40 nucleotides in length.

10. A method according to claim 1 wherein the nucleoside triphosphates of paragraph (c) include at least two deoxynucleotides, at least one of which deoxynucleotide comprises a detectable label or an attachment moiety capable of binding a detectable label.

11. A method according to either of claims 1 or 2 wherein each deoxynucleotide of the nucleoside triphosphates of paragraph (c) is a deoxyribonucleoside triphosphate selected from the group consisting of dATP, dGTP, dCTP, dUTP, and dTTP.

12. A method according to claim 1 wherein each deoxynucleotide of the nucleoside triphosphates of paragraph (c) is a deoxyribonucleoside triphosphate selected from the group consisting of dATP, dGTP, dCTP, dUTP, and dTTP and in which the detectable label is a radioisotope.

13. A method according to either of claims 1 or 2 wherein each chain-terminating nucleotide analogue of the nucleoside triphosphates of paragraph (c) is a dideoxyribonucleotide selected from the group consisting of ddATP, ddGTP, ddCTP, and ddTTP.

15. A method according to either of claims 1 or 2, wherein the polymerization agent is a DNA polymerase.

16. A method according to either of claims 1 or 2, wherein the primer extension product is removed from the target nucleic acid prior to analysis.

2. A method for detecting the presence of one or more specific nucleotides at a predetermined target position in a target nucleic acid, the method comprising the steps of:

(a) providing an analyzable amount of the target nucleic acid in a single stranded form;

(b) hybridizing the target nucleic acid with a detection primer to form a target-nucleic-acid/detection-primer hybrid, the detection primer comprising a detection-primer nucleotide sequence having a primer-extension-initiation 3′

-end nucleotide which constitutes a 3′

end of the detection primer, the detection-primer nucleotide sequence being complementary to a primer-hybridizing nucleotide sequence of the target nucleic acid with a nucleotide in the target nucleic acid complementary to the primer-extension-initiation 3′

-end nucleotide of the detection-primer nucleotide sequence defining a primer-end complement nucleotide, the primer-hybridizing nucleotide sequence of the target nucleic acid extending towards the 3′

end of the target nucleic acid from the primer-end complement nucleotide, the primer-end complement nucleotide being located in the target nucleic acid at a position 3′

-ward of the predetermined target position, the position of the primer-end complement nucleotide being subject to a constraint that no nucleotide of the same type as the one or more specific nucleotides to be detected be located in the target nucleic acid in any position between the position of the primer-end complement nucleotide and the predetermined target position;

(c) forming an extension-reaction mixture by exposing the target-nucleic-acid/detection-primer hybrid to an admixture of a polymerization agent and a plurality of nucleoside triphosphates, the nucleoside triphosphates of the admixture including at least one deoxynucleotide and at least one chain-terminating nucleotide analogue, at least one chain-terminating nucleotide analogue defining a labeled chain-terminating nucleotide analogue comprising a detectable label or an attachment moiety capable of binding a detectable label, each deoxynucleotide of the admixture of nucleoside triphosphates being complementary to a nucleotide which differs from any nucleotide to which a chain-terminating nucleotide analogue of the admixture is complementary, the plurality of nucleoside triphosphates of the admixture being such that, if a labeled chain-terminating nucleotide analogue is complementary to a specific nucleotide at the predetermined target position, a detectable primer-extension product is formed of the detection primer extended to include an extension portion terminated with the labeled chain-terminating nucleotide analogue; and

(d) analyzing the extension-reaction mixture from step (c) for the presence or absence of a detectable label in association with a labeled chain-terminating nucleotide analogue terminating an extension portion of a primer extension product to detect the presence of the corresponding specific nucleotide at the target position in the target nucleic acid.

14. A method according to claim 2 wherein each chain-terminating nucleotide analogue of the nucleoside triphosphates of paragraph (c) is a dideoxyribonucleotide selected from the group consisting of ddATP, ddGTP, ddCTP, and ddTTP and in which the detectable label is a fluorescent group.