Nucleic acid detection methods using universal priming

• US 7,361,488 B2
• Filed: 08/09/2002
• Issued: 04/22/2008
• Est. Priority Date: 02/07/2000
• Status: Expired due to Fees

• 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): LU, FRANK WEI MIN
• Application Number:
  US10/215,644
• Publication Number:
  US 20030104434A1
• Time in Patent Office: 2,083 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.51, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 436/501, 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 with a minimum or absence of target-specific amplification.

Claims

1. A method of detecting a splice junction sequence in a plurality of target pre-mRNA sequences, said method comprising:

a) hybridizing said plurality of target pre-mRNA sequences to a plurality of first splice junction specific probes and forming first hybrids wherein each of said plurality of target pre-mRNA sequences comprises a first target domain, a second target domain and a poly(A) sequence, said first target domain and said second target domain are adjacent each other and said first target domain comprises a splice junction sequence, and each of said plurality of first splice junction specific probes comprises;

i) an upstream universal priming site (UUP), wherein each of said first splice junction specific probes has the same UUP;

ii) at least one adapter; and

iii) a first target-specific sequence substantially complementary to said first target domain, and said upstream universal priming site (UUP) locates in the upstream of said adapter and said first target-specific sequence;

b) hybridizing said plurality of target pre-mRNA sequences of said first hybrids to a plurality of second probes and forming second hybrids wherein each of said second hybrids comprises a said target pre-mRNA sequence, a said first splice junction specific probe and a said second probe and each of said plurality of second probes comprises;

iv) a second target-specific sequence substantially complementary to said second target domain;

v) a downstream universal priming site (DUP), wherein each of said second probes has the same DUP, and said downstream universal priming site (DUP) locates in the downstream of said second target-specific sequence, and wherein, in each of said plurality of first and second hybrids, said poly(A) sequence remains single-stranded;

c) contacting said second hybrids with a ligase and forming ligation complexes comprising ligated probes, each of said ligated probes comprising a said first splice junction specific probe and a said second probe;

d) contacting said ligation complexes with a support comprising a poly(T) sequence, such that said poly(A) sequence hybridizes with said poly(T) sequence;

e) removing unhybridized said first splice junction specific probes and unhybridized said second probes;

f) denaturing said ligation complexes;

g) amplifying the ligated probes using universal primers that hybridize to the UUP and DUP, or complement thereof, and generating a plurality of amplicons each comprising a said adapter;

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

i) detecting said assay complexes wherein the detection of said assay complexes is an indication of the presence of said splice junction sequence in the pre-mRNA sequences.

2. The 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 second hybrids with a polymerase and at least one dNTP.

3. The method according to claim 1 wherein one of said first splice junction probe and said second probes comprises a label.

4. The method according to claim 3 wherein said label is a primary label.

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

6. The method according to claim 1 wherein said universal primers comprise a first primer and a second primer and said amplifying in method step g) is done by:

a) hybridizing said first universal primer to said UUP;

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

c) hybridizing said 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).

7. The method according to claim 1 wherein said capture probes comprise a first capture probe and a second capture probe and said array in method step h) 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 said first capture probe and a second subpopulation comprising a said second capture probe.

8. The method according to claim 7 wherein said discrete sites comprises wells.

9. The method according to claim 7 wherein said substrate comprises a fiber optic bundle.

10. The method according to claim 1 wherein said support comprising a poly(T) sequence comprises magnetic beads.

11. The method according to claim 1, wherein said capture probes comprise a first capture probe and a second capture probe and said array in method step h) comprises a population of microspheres comprising at least a first subpopulation comprising a said first capture probe and a second subpopulation comprising a said second capture probe.

12. The method according to claim 11, wherein said microspheres are in a solution.

13. A method of detecting a splice junction sequence in a target pre-mRNA sequence, said method comprising:

a) hybridizing said target pre-mRNA sequence to a first splice junction specific probe and forming a first hybrid wherein said target pre-mRNA sequence comprises a first target domain, a second target domain and a poly(A) sequence, said first target domain and said second target domain are adjacent each other and said first target domain comprises a splice junction sequence and said first splice junction specific probe comprises;

i) an upstream universal priming site (UUP);

ii) at least one adapter; and

iii) a first target-specific sequence substantially complementary to said first target domain, wherein said upstream universal priming site (UUP) locates in the upstream of said adapter and said first target-specific sequence, and said adapter is exogenous to said first target-specific sequence;

b) hybridizing said target pre-mRNA sequence of said first hybrid to a sequence of a second probe and forming a second hybrid wherein said second hybrid comprises a said target pre-mRNA sequence, a said first splice junction specific probe and a said second probe and said second probe comprises;

iv) a second target-specific sequence substantially complementary to said second target domain;

v) a downstream universal priming site (DUP), wherein said downstream universal priming site (DUP) locates in the downstream of said second target-specific sequence, and said poly(A) sequence remains single-stranded in each of said first and second hybrids;

c) contacting said second hybrid with a ligase and forming a ligation complex comprising ligated probes, each of said ligated probes comprising a said first splice junction specific probe and a said second probe;

d) contacting said ligation complex with a support comprising a poly(T) sequence, such that said poly(A) sequence hybridizes with said poly(T) sequence;

e) removing unhybridized said first splice junction specific probe and unhybridized said second probe;

f) denaturing said ligation complex;

g) amplifying the ligated probes using universal primers that hybridize to the UUP and DUP, or complement thereof, and generating a plurality of amplicons each comprising a said adapter;

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

i) detecting said assay complexes wherein the detection of said assay complexes is an indication of the presence of said splice junction sequence in the pre-mRNA sequence.

14. A method of detecting a splice junction sequence in a plurality of target pre-mRNA sequences, said method comprising:

a) hybridizing said plurality of target pre-mRNA sequences to aplurality of first splice junction specific probes and form first hybrids wherein each of said plurality of target pre-mRNA sequences comprises a first target domain, a second target domain and a poly(A) sequence, said first target domain and said second target domain are adjacent each other and said first target domain comprises a splice junction sequence; and

each of said plurality of first splice junction specific probes comprises;

i) an upstream universal priming site (UUP), wherein each of said first splice junction specific probes has the same UUP;

ii) at least one adapter; and

iii) a first target-specific sequence substantially complementary to said first target domain, and said upstream universal priming site (UUP) locates in the upstream of said adapter and said first target-specific sequence;

b) hybridizing said plurality of target pre-mRNA sequences of said first hybrids to a plurality of second probes and forming second hybrids wherein each of said second hybrids comprises a said target pre-mRNA sequence, a said first splice junction specific probe and a said second probe and each of said plurality of second probes comprises;

iv) a second target-specific sequence substantially complementary to said second target domain;

v) a downstream universal priming site (DUP), wherein each of said second probes has the same DUP, and said downstream universal priming site (DUP) locates in the downstream of said second target-specific sequence;

c) hybridizing said second hybrids to a poly(T) sequence on a solid support and forming third hybrids, such that said poly(A) sequence hybridizes with said poly(T) sequence;

d) contacting said third hybrids with a ligase and forming ligation complexes comprising ligated probes, each of said ligated probes comprising a said first splice junction specific probe and a said second probe;

e) removing unhybridized said first splice junction specific probes and unhybridized said second probes;

f) denaturing said ligation complexes;

g) amplifying the ligated probes using universal primers that hybridize to the UUP and DUP, or complement thereof, and generating a plurality of amplicons each comprising a said adapter;

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

i) detecting said assay complexes wherein the detection of said assay complexes is an indication of the presence of said splice junction sequence in the pre-mRNA sequences.