Length determination of nucleic acid repeat sequences by discontinuous primer extension

• US 5,945,284 A
• Filed: 05/27/1997
• Issued: 08/31/1999
• Est. Priority Date: 05/27/1997
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Livak, Kenneth J., Blasband, Andrew J., Lowe, Adam L.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/863,437
• Time in Patent Office: 826 Days
• Field of Search: 435/6, 935/76, 935/77, 935/78, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

In a first aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer extension reagent; separating the target-primer hybrid and unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent; measuring a signal produced by the label; treating the label so as to render the label undetectable; and repeating the above steps until the signal is substantially less than a signal detected in a previous cycle. In a second aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer-extension reagent; separating the target-primer hybrid from unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent and with a primer termination reagent, the primer termination reagent including a nucleotide terminator having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent and unreacted primer termination reagent; measuring a signal produced by the label; and repeating the above steps until a signal is detected indicating incorporation of the nucleotide terminator. The invention further includes kits useful for practicing the above methods.

Claims

1. A method for determining the number of repeat units in a repeat region of a target nucleic acid comprising the steps of:

(a) annealing a primer-complementary portion of a target nucleic acid to a primer thereby forming a target-primer hybrid;

(b) performing a first primer extension reaction using a first primer extension reagent;

(c) separating the target-primer hybrid and unreacted first primer extension reagent;

(d) performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto;

(e) separating the target-primer hybrid from unreacted second primer extension reagent;

(f) measuring a signal produced by the label;

(g) treating the label so as to render the label undetectable;

(h) repeating a cycle of steps (a) through (g) until the signal is substantially less than a signal detected in a previous cycle; and

(i) determining a number of repeat units in a repeat region of the target nucleic acid.

2. The method of claim 1 wherein step (d) further includes reacting the target-primer hybrid with a primer termination reagent.

3. The method of claim 1 wherein the target-primer hybrid is attached to a solid support.

4. The method of claim 1 wherein the primer is attached to a solid support.

5. The method of claim 1 wherein the target nucleic acid is attached to a solid support.

6. The method of claim 1 wherein the label is selected from the group consisting of fluorescent and chemiluminescent molecules.

7. The method of claim 1 wherein the label is attached to the extendible nucleotide through a cleavable linker.

8. The method of claim 1 wherein the target nucleic acid is amplified prior to analysis.

9. The method of claim 8 wherein amplification is achieved using a PCR.

10. The method of claim 1 wherein the step of treating the label so as to render the label undetectable includes cleaving the label from the labeled extendible nucleotide.

11. The method of claim 1 wherein the step of treating the label so as to render the label undetectable includes destroying a signal producing property of the label.

12. A kit useful for determining the number of repeat units in a repeat region of a target nucleic acid comprising:

a primer having a sequence complementary to a primer-complementary portion of a target nucleic acid;

a first primer extension reagent comprising a set of extendible nucleotides that allow a primer extension reaction to proceed only to the extent that a primer is extended by an amount less than a full repeat unit; and

a second primer extension reagent comprising a set of extendible nucleotides that allow a primer extension reaction to proceed only to the extent that the portion of a repeat unit not synthesized by the first primer extension reagent is synthesized;

wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto.

13. The kit of claim 12 wherein the primer is attached to a solid support.

14. The kit of claim 12 wherein the label is selected from the group consisting of fluorescent and chemiluminescent molecules.

15. The kit of claim 12 wherein the label is attached to the extendible nucleotide through a cleavable linker.