Detection of nucleic acids by target-specific hybrid capture method

• US 20060051809A1
• Filed: 11/07/2005
• Published: 03/09/2006
• Est. Priority Date: 06/15/2000
• Status: Active Grant

• Current Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Original Assignee: QIAGEN Gaithersburg, Inc. (Qiagen NV)
• Inventors: Lowe, Brian, Nazarenko, Irina, Lorincz, Attila, Thai, Ha, Eder, Paul, Mallonee, Richard
• Granted Patent: US 7,601,497 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/119 Triple helix formation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

Target-specific hybrid capture (TSHC) provides a nucleic acid detection method that is not only rapid and sensitive, but is also highly specific and capable of discriminating highly homologous nucleic acid target sequences. The method produces DNA:RNA hybrids which can be detected by a variety of methods.

Claims

1. A method of detecting each of a plurality of target nucleic acids comprising:

a) capturing a plurality of target nucleic acids onto a solid support by mixing;

said plurality of target nucleic acids, nucleic acid probes complementary to at least a portion of the target nucleic acids, wherein one is RNA and the other is DNA, and a solid support, forming a plurality of enriched target nucleic acids;

b) amplifying the enriched nucleic acids or nucleic acid probes, forming a plurality of amplified targets; and

c) detecting the target nucleic acids by detection of the plurality of amplified targets by mixing;

the plurality of amplified targets;

selectable oligonucleotides which hybridize to a first portion of the amplified targets; and

nucleic acid probes complementary to a second portion of the amplified targets, forming a DNA;

RNA hybrid oligonucleotide complex, wherein the DNA;

RNA hybrids are detectable by DNA;

RNA hybrid-specific binding agents, and selectable by the oligonucleotide complex separation, thereby detecting each of the plurality of target nucleic acids.

2. The method according to claim 1, wherein in the capturing step, the solid support is conjugated to a DNA:

RNA hybrid-specific binding agent, thereby capturing the target nucleic acids to a solid support.

3. The method according to claim 1, wherein the DNA:

RNA hybrid-specific binding agent is selected from the group consisting of;

DNA;

RNA hybrid-specific antibodies, monoclonal or polyclonal antibodies, or fragments thereof, proteins, catalytically inactive RNase H, and nucleic acids, nucleic acid aptamers, or oligonucleotides which bind and form a triplex structure.

4. The method according to claim 2, wherein the DNA:

RNA hybrid-specific binding agent is a DNA;

RNA hybrid-specific antibody.

5. The method according to claim 2, wherein the DNA:

RNA hybrid-specific binding agent is a nucleic acid probe complementary to the target nucleic acid.

6. The method according to claim 3, wherein the DNA:

RNA hybrid-specific binding agent is detectably labeled.

7. The method according to claim 6, wherein the label is detected by:

colorimetry, chemiluminescence, fluorescence, and light-scattering methods.

8. The method according to claim 1, wherein the captured target nucleic acids or nucleic acid probes are amplified by isothermal amplification.

9. The method according to claim 8, wherein DNA polymerase is used in the isothermal amplification.

10. The method according to claim 8, wherein random primers are used in the isothermal amplification.

11. The method according to claim 10, wherein the random primers are pentamers.

12. The method according to claim 10, wherein the random primers contain a subset of primer sequences specific for a disease.

13. The method according to claim 1, wherein the selectable oligonucleotides are bound to a solid support.

14. The method according to claim 1, wherein the solid support is selected from the group consisting of:

plates, microplates, microtiter plates, slides, dishes, beads, particles, microparticles, cups, strands, chips, microchips, strips, membranes, microarrays, and test tubes.

15. The method according to claim 14, wherein the solid support is made of material selected from the group consisting of:

glass, silicon, plastic, polystyrene, polyethylene, polypropylene, and polycarbonate.

16. The method according to claim 14, wherein the solid support is modified to contain:

has been modified so that the surface contains carboxyl, amino, hydrazide, aldehyde groups, nucleic acid or nucleotide derivatives, primary amino groups, and dyes.

17. The method according to claim 1, further comprising adding blocker probes.

22. The method of claim 1, wherein the nucleic acid probes complementary to at least a portion of the target nucleic acids and oligonucleotide sequences are selected from the group consisting of:

23. A composition for use in the method of claim 17, wherein the the nucleic acid probes complementary to at least a portion of the target nucleic acids, oligonucleotide sequences, and blocker probes are selected from the group consisting of:

18. A method of detecting each of a plurality of target nucleic acids comprising:

a) hybridizing a plurality of target nucleic acids to nucleic acid probes which are complementary to at least a portion of the target nucleic acids, forming DNA;

RNA hybrids;

b) capturing the DNA;

RNA hybrids with DNA;

RNA hybrid-specific antibodies conjugated to solid supports;

c) separating unbound target nucleic acids from the DNA;

RNA hybrids;

d) amplifying the captured target nucleic acids or nucleic acid probes, forming a plurality of amplified targets, using random primers and DNA polymerase;

e) hybridizing nucleic acid probes complementary to a first portion of the amplified targets, forming DNA;

RNA hybrids;

f) hybridizing oligonucleotides conjugated to a plurality of solid supports to a second portion of the amplified targets, wherein the solid support is selectable; and

g) selecting each of the plurality of amplified targets by the selectable solid support; and

h) detecting each of the plurality of amplified targets by binding DNA;

RNA hybrid-specific binding agents to the DNA;

RNA hybrids, wherein the presence of the plurality of amplicons is indicative of the presence of the target nucleic acids.

19. The method according to claim 18, wherein the solid support conjugated to oligonucleotides are distinguishable beads.

20. The method according to claim 19, wherein the distinguishable beads are selected from a group consisting of detectable labels:

a fluorescent label, a gold label, and an enzyme label.

21. A multiplex method of detecting a plurality of target DNAs comprising:

a) hybridizing a plurality of target DNAs to RNA probes which are complementary to at least a portion of the target DNAs, forming DNA;

RNA hybrids;

b) capturing the DNA;

RNA hybrids with DNA;

RNA hybrid-specific antibodies conjugated to beads;

c) separating unbound nucleic acids from DNA;

RNA hybrids, forming enriched targets;

d) amplifying the enriched target DNAs, forming a plurality of amplified targets, using random primers and DNA polymerase;

e) hybridizing RNA probes complementary to a first portion of the amplified target DNAs, forming DNA;

RNA hybrids;

f) hybridizing DNA oligonucleotides to a second portion of the target DNAs, wherein the DNA oligonucleotides are conjugated to a plurality of selectable beads; and

g) detecting the plurality of amplified target DNAs by binding DNA;

RNA hybrid-specific antibodies to the DNA;

RNA hybrids and selecting each of the amplified targets based on the particular selectable bead.

24. A composition of matter comprising a nucleic acid sequence selected from the group consisting of:

SEQ ID NOs;

1-124.