Length determination of nucleic acid repeat sequences by discontinuous primer extension

• US 6,773,887 B2
• Filed: 10/22/2001
• Issued: 08/10/2004
• Est. Priority Date: 05/27/1997
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Livak, Kenneth J., Blasband, Andrew J., Lowe, Adam L.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US10/038,520
• Publication Number:
  US 20040126756A1
• Time in Patent Office: 1,023 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays

Abstract

Disclosed is a method for determining the number of repeat units in a repeat region of a target nucleic acid. 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, said method 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, wherein the first primer extension reagent allows said first primer extension reaction to proceed only to the extent that said primer is extended by an amount less than a full repeat unit;

(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 the second primer extension reagent allows said second primer extension reaction to proceed only to the extent that the portion of said full repeat unit not synthesized by the first primer extension reagent is synthesized and 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 the number of repeat units in said repeat region of said target nucleic acid.

2. The method of claim 1, wherein step (d) further includes reacting the target-primer hybrid with a primer termination reagent, wherein the termination reagent includes one or more nucleotide terminators that prevent the further extension of a primer extension product when incorporated into said primer extension product.

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 method for determining the number of repeat units in a repeat region of a target nucleic acid, said method 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, wherein the first primer extension reagent allows said first primer extension reaction to proceed only to the extent that said primer is extended by an amount less than a full repeat unit;

(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 the second primer extension reagent allows said second primer extension reaction to proceed only to the extent that the portion of said full repeat unit not synthesized by the first primer extension reagent is synthesized and a primer termination reagent including a nucleotide terminator having a label attached reagent;

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

(f) measuring a signal produced by the label;

(g) repeating a cycle of steps (a) through (f) until a signal is detected indicating incorporation of the nucleotide terminator; and

(h) determining the number of repeat units in said repeat region of said target nucleic acid.

13. The method of claim 12, wherein the target-primer hybrid is attached to a solid support.

14. The method of claim 12, wherein the primer is attached to a solid support.

15. The method of claim 12, wherein the target nucleic acid is attached to a solid support.

16. The method of claim 12, wherein the label is selected from the group consisting of fluorescent and chemiluminescent molecules.

17. The method of claim 12, wherein the target nucleic acid is amplified prior to analysis.

18. The method of claim 17, wherein amplification is achieved using a PCR.