Nucleic acid detection methods using universal priming

• US 6,812,005 B2
• Filed: 02/07/2001
• Issued: 11/02/2004
• Est. Priority Date: 02/07/2000
• Status: Expired due to Term

• Current Assignee: Illumina Incorporated, Regents of the University of California (University of California)
• Original Assignee: Illumina Incorporated, Regents of the University of California (University of California)
• Inventors: Fan, Jian-Bing, Fu, Xiang-Dong
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/779,202
• Publication Number:
  US 20020172946A1
• Time in Patent Office: 1,364 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention is directed to providing sensitive and accurate assays for gene detection, genome-wide gene expression profiling and alternative splice monitoring wit a minimum or absence of target-specific amplification.

Claims

1. A method of detecting a first target sequence comprising a first target domain, a second adjacent target domain and a single stranded poly(A) sequence, said method comprising:

a) hybridizing a first probe to said first target domain comprising;

i) an upstream universal priming site (UUP); and

ii)) a first target-specific sequence substantially complementary to said first target domain;

b) hybridizing a second probe to said second probe to said second adjacent target domain comprising;

iii) a second target-specific sequence substantially complementary to second adjacent target domain;

iv) a downstream universal priming site (DUP);

 

wherein at least one of said first and second probe comprises at least a first adapter sequence;

, said poly(A) sequence remains single-stranded, and wherein said target sequence and said first and second probes form a litigation complex;

c) contacting said ligated complex with a ligase to form a ligated complex;

d) contacting said ligated complex with a support having a poly(T) sequence, such that said single stranded poly(A) sequence hybridizes with said poly(T) sequence;

e) removing unhybridized first and second probe sequence;

f) denaturing said ligation complex;

g) amplifying the ligated first and second probes to generate a plurality of amplicons;

h) contacting said amplicons with an array of capture probes to form assay complexes; and

detecting said assay complexes.

2. A method according to claim 1 wherein said first target domain and said second target domain are directly adjacent.

3. A method according to claim 1 wherein said first target domain and said second target domain are separated by at least one base and said method further includes contacting said ligation complex with a polymerase and at least one dNTP.

4. A method according to claim 1, 2 or 3 wherein one of said first and second probes comprises a label.

5. A method according to claim 4 wherein said label is a primary label.

6. A method according to claim 5 wherein said label is a fluorescent label.

7. A method according to claim 4 wherein said label is a secondary label.

9. A method according to claim 1, 2 or 3 wherein said amplifying is done by:

a) hybridizing a first universal primer to said UUP;

b) providing a polymerase and dNTPs such that said first universal primer is extended;

c) hybridizing a second universal primer to said DUP;

d) providing a polymerase and dNTPs such that said second universal primer is extended; and

e) repeating steps a) through d).

10. A method according to claim 1, 2 or 3 said array comprises:

a) a substrate with a patterned surface comprising discrete sites; and

b) a population of microspheres comprising at least a first subpopulation comprising a first capture probe and a second subpopulation comprising a second capture probe.

11. A method according to claim 10 wherein said discrete sites comprise wells.

12. A method according to claim 10 wherein said substrate comprises a fiber optic bundle.

13. A method according to claim 1, 2 or 3 wherein said support magnetic beads comprising a poly(T) sequence.

14. A method according to claim 9 wherein at least one of said first universal primers and said second universal primer comprises a label.

15. A method according to claim 14 wherein said label is a primary label.

16. A method according to claim 15 wherein said label is a fluorescent label.

17. A method according to claim 14 wherein said label is a secondary label.

18. A method according to claim 17 wherein said secondary label is biotin.

19. A method according to claim 9 wherein said dNTPs comprise a label.

20. A method according to claim 19 wherein said label is a primary label.

21. A method according to claim 20 wherein said label is a fluorescent label.

22. A method according to claim 19 wherein said label is a secondary label.

23. A method according to claim 22 wherein said secondary label is biotin.

8. A method according to claim 8 wherein said secondary label is biotin.