Microarray-based analysis of polynucleotide sequence variations

• US 6,376,191 B1
• Filed: 11/06/2000
• Issued: 04/23/2002
• Est. Priority Date: 03/22/2000
• Status: Expired due to Fees

• Current Assignee: Mergen Ltd.
• Original Assignee: Mergen Ltd.
• Inventors: Hu, Qianjin, Yu, Zailin, Peng, Zaoyuan
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/707,366
• Time in Patent Office: 533 Days
• Field of Search: 435/6, 435/91.2, 435/810, 536/24.33
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Solid phase polymerase-mediated amplification approaches using immobilized primers on a microarray are provided for detecting sequence variations in a target polynucleotide. The methods and compositions provided herein are useful for research and clinical applications, particularly for large scale assays of genetic information in biological samples of interest.

Claims

1. A method for identifying sequence variations in a target polynucleotide in a sample as compared to a reference sequence, the method comprising the steps of:

a) contacting an array of one or more groups of oligonucleotide primers with a reaction mixture containing the sample and reagents for polymerase-mediated polynucleotide amplification, wherein the array of oligonucleotide primers is immobilized on a solid phase support by the 5′

-ends of the oligonucleotides, and further wherein each group of oligonucleotide primers is selected to span a particular region of the reference sequence, occupy a discrete area on the array, and comprise at least two sets of primers;

     1) a first set that is exactly complementary to the reference sequence; and

     2) one or more additional sets, each of which is identical to the first set of primers but for the nucleotide at the 3′

-end which is different in each additional set;

b) performing a plurality of cycles of polymerase-mediated polynucleotide amplification, whereby the target polynucleotide serves as template for the synthesis of detectable nascent polynucleotides which are extended from the sets of primers that are exactly complementary to the target polynucleotide, wherein all of the oligonucleotide primers of the array are present in the same amplification reaction;

c) detecting the presence of synthesized polynucleotides which are captured on discrete areas of the solid phase support via corresponding immobilized primers; and

d) identifying sequence variations in the at least one target polynucleotide according to a detected pattern of synthesized polynucleotides on the solid phase support.

2. The method of claim 1, wherein the reaction mixture further comprises a population of solution phase primers.

3. The method of claim 2, wherein the population of solution phase primers includes an universal primer.

4. The method of claim 3, wherein the universal primer is an oligo-dT primer.

5. The method of claim 3, wherein the universal primer is a primer containing a T7 promoter, a T3 promoter or an SP6 promoter.

6. The method of claim 2, wherein the population of solution phase primers includes multiple primers each having specific sequences.

7. The method of claim 1, wherein sequence variations in least two different target polynucleotides in a sample are identified.

8. The method of claim 1, wherein the reaction mixture comprises a detectable label that is incorporated into the nascent polynucleotides, thereby making the nascent polynucleotides detectable.

9. The method of claim 8, wherein the detectable label is a fluorescent molecule.

10. The method of claim 8, wherein the detectable label is at least one of radiolabeled-dNTP, fluoro-dNTP, biotinylated dNTP or a digoxigenin-dNTP.

11. The method of claim 8, wherein the detectable label is conjugated to a molecule that binds the nascent polynucleotide.

12. The method of claim 11 wherein the detectable label is conjugated to a molecule that binds a second label incorporated into the nascent polynucleotide.

13. The method of claim 1 which is a method for identifying sequence variations in at least one target polynucleotide in a sample as compared to at least one reference sequence, wherein the array comprises from at least about 100 to at least about 100,000 groups of immobilized oligonucleotide primers, and wherein each group of oligonucleotide primers is selected to span a particular region of one of the at least one reference sequences.

14. The method of claim 13, wherein the array comprises at least about 1,000 groups of immobilized oligonucleotide primers.

15. The method of claim 14, wherein the array comprises at least about 10,000 groups of immobilized oligonucleotide primers.

16. The method of claim 1, wherein the solid phase support is made of material selected from the group consisting of glass, plastics, synthetic polymers, ceramic and nylons.

17. The method of claim 1, wherein the polymerase is Taq polymerase, TthI polymerase, Vent polymerase, Pfu polymerase or any other thermostable polymerase.

27. The method of claim 1, wherein each group of oligonucleotide primers of the array comprises four sets of primers:

1) a first set that is exactly complementary to the reference sequence; and

     2) three additional sets, each of which is identical to the first set of primers but for the nucleotide at the 3′

-end which is different in each additional set.

18. A kit for identifying sequence variations in a target polynucleotide as compared to a reference sequence, comprising:

a) an array of multiple groups of oligonucleotide primers immobilized to a solid phase support, wherein each group of oligonucleotide primers is selected to span a particular region of the reference sequence, occupying a discrete area of the array, and comprising at least four sets of primers;

     1) a first set that is exactly complementary to the reference sequence; and

     2) three additional sets of primers, each of which is identical to the first set of primers but for the nucleotide at the 3′

-end, which is different in each of the three sets;

b) reagents suitable for a polymerase-mediated polynucleotide amplification reaction on the array; and

c) detection means for detecting amplified polynucleotides on the array.

19. The kit of claim 18, wherein the detection means comprises a detectable label that is incorporated into the amplified polynucleotides during the amplification reaction.

20. The kit of claim 19, wherein the detectable label is a fluorescent molecule.

21. The kit of claim 19, wherein the detectable label is a biotinylated 11-dNTP or a digoxigenin-dNTP.

22. An array for identifying sequence variations in at least one target polynucleotide in a sample as compared to at least one reference sequence by polymerase-mediated polynucleotide amplification, the array comprising:

about 100 to about 100,000 groups of oligonucleotide primers immobilized on discrete areas of a solid phase support, wherein the group of oligonucleotide primers is selected to span a particular region of one of the at least one reference sequences, each group comprising;

at least two sets of primers wherein

     1) a first set is exactly complementary to the reference sequence, and

     2) each of one or more additional sets is identical to the first set of primers except for the nucleotide at the 3′

-end which is different in each additional set, such that the target polynucleotide serves as template for the synthesis of detectable nascent polynucleotides extended from the sets of primers that are exactly complementary to the target polynucleotide.

23. The array of claim 22, wherein the array comprises at least about 1,000 groups of immobilized oligonucleotide primers.

24. The array of claim 23, wherein the array comprises at least about 10,000 groups of immobilized oligonucleotide primers.

25. The array of claim 22, wherein the solid phase support is made of material selected from the group consisting of glass, plastics, synthetic polymers, ceramic and nylons.

26. The array of claim 22, wherein the array of oligonucleotide primers is suitable for identifying sequence variations in two or more target polynucleotides.

28. A kit for identifying sequence variations in at least one target polynucleotide as compared to at least one reference sequence, comprising:

a) an array of from about 100 to about 100,000 groups of oligonucleotide primers immobilized to a solid phase support, wherein each group of oligonucleotide primers is selected to span a particular region of one of the at least one reference sequence, occupying a discrete area of the array, and comprising at least two sets of primers;

     1) a first set that is exactly complementary to the reference sequence; and

     2) one or more additional sets of primers, each of which is identical to the first set of primers but for the nucleotide at the 3′

-end, which is different in each additional set;

b) reagents suitable for a polymerase-mediated polynucleotide amplification reaction on the array; and

c) detection means for detecting amplified polynucleotides on the array.

29. A kit for identifying sequence variations in a target polynucleotide as compared to a reference sequence, comprising:

a) an array of multiple groups of oligonucleotide primers immobilized to a solid phase support, wherein each group of oligonucleotide primers is selected to span a particular region of the reference sequence, occupying a discrete area of the array, and comprising at least two sets of primers;

     1) a first set that is exactly complementary to the reference sequence; and

     2) one or more additional sets of primers, each of which is identical to the first set of primers but for the nucleotide at the 3′

-end, which is different in each additional set;

b) reagents suitable for polymerase-mediated polynucleotide amplification on the array of both strands of the target polynucleotide, wherein the reagents comprise at least one solution-phase oligonucleotide primer; and

c) detection means for detecting amplified polynucleotides on the array.

30. An array for identifying sequence variations in a target polynucleotide in a sample as compared to a reference sequence by polymerase-mediated polynucleotide amplification, the array comprising:

one or more groups of oligonucleotide primers immobilized on discrete areas of a solid phase support, wherein the group of oligonucleotide primers is selected to span a particular region of the reference sequence, each group comprising;

four sets of primers wherein

     1) a first set is exactly complementary to the reference sequence, and

     2) each of three additional sets is identical to the first set of primers except for the nucleotide at the 3′

-end which is different in each additional set, such that the target polynucleotide serves as template for the synthesis of detectable nascent polynucleotides extended from the sets of primers that are exactly complementary to the target polynucleotide.