T-structure invasive cleavage assays, consistent nucleic acid dispensing, and low level target nucleic acid detection

• US 20080131870A1
• Filed: 06/11/2007
• Published: 06/05/2008
• Est. Priority Date: 06/09/2006
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Third Wave Technologies Incorporated (Hologic Incorporated)
• Inventors: Chehak, LuAnne, Wu, Kwok, Allawi, Hatim Taysir, Lyamichev, Victor A., Elagin, Vecheslav A., Law, Scott M., Fleming, Chris, Iszczyszyn, Walter
• Granted Patent: US 7,759,062 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

The present invention relates to systems, methods and kits for low-level detection of nucleic acids, detecting at least two different viral sequences in a single reaction vessel, and increasing the dynamic range of detection of a viral target nucleic acid in a sample. The present invention also relates to T-structure invasive cleavage assays, as well as T-structure related target dependent non-target amplification methods and compositions. The present invention further relates to methods, compositions, devices and systems for consistent nucleic acid dispensing onto surfaces.

Claims

1. A method for detecting target nucleic acid in a sample, wherein the number of copies of said target nucleic acid initially present in said sample is suspected to contain between about 2 and about 100 copies, wherein said method comprises;

a) incubating said sample with a plurality of first and second probes, a plurality of reporter sequences, and a cleavage agent under conditions such that;

i) said first probes hybridize to first regions of said target nucleic and are cleaved by said cleavage agent thereby generating first 5′

cleaved portions, and

ii) said second probes hybridize to second regions of said target nucleic acid and are cleaved by said cleavage agent thereby generating second 5′

cleaved portions; and

b) detecting a signal from said reporter sequences generated by hybridization of said first and second 5′

cleaved portions to said reporter sequences thereby detecting the presence of said target nucleic acids in said sample.

2. The method of claim 1, wherein said first and second regions of said target nucleic acid are amplified prior to step a).

3. The method of claim 1, wherein said first and second regions of said target nucleic acid are amplified with at least 30 rounds of PCR prior to step a).

4. The method of claim 1, wherein said target nucleic acid is viral nucleic acid.

5. The method of claim 4, wherein said viral nucleic acid is from hepatitis C virus.

6. The method of claim 1, wherein said signal is generated by cleavage of said reporter sequences.

7. The method of claim 1, wherein said first and second probes are cleaved as part of an invasive cleavage reaction.

8. A method for detecting the presence or absence of a target nucleic acid in a sample comprising;

a) incubating said sample with a stem oligonucleotide, an upstream oligonucleotide, a downstream probe, and a cleavage agent under conditions such that, if said target nucleic acid is present;

i) a 3′

target specific region of said stem oligonucleotide hybridizes to said target nucleic acid, and a stem region of said stem oligonucleotide remains available for hybridization to a portion of said upstream oligonucleotide and said downstream probe,

ii) a 5′

target specific region of said upstream oligonucleotide hybridizes to said target nucleic acid, and a stem specific region of said upstream oligonucleotide hybridizes to a portion of said stem region of said stem oligonucleotide,

iii) a 3′

region of said downstream probe hybridizes to a portion of said stem region of said stem oligonucleotide, and a 5′

region of said downstream probe does not hybridize to said stem region of said stem oligonucleotide, wherein said downstream probe forms an invasive cleavage structure with said upstream oligonucleotide and said stem region of said stem oligonucleotide, and

iv) said cleavage agent cleaves said downstream probe in said invasive cleavage structure thereby generating a 5′

cleaved portion; and

b) detecting the presence or absence of said target nucleic acid in said sample.

9. The method of claim 8, wherein said detecting comprises determining if said 5′

cleaved portion has been generated.

10. The method of claim 8, wherein said incubating further includes a reporter sequence, and said detecting the presence or absence of said target nucleic acid comprises detecting a signal from said reporter sequence generated by hybridization of said 5′

cleaved portion to said reporter sequence.

11. The method of claim 10, wherein said hybridization of said 5′

cleaved portion to said reporter sequence generates an invasive cleavage structure that is cleaved by said cleavage agent.

12. The method of claim 11, wherein said target nucleic acid is composed of RNA and said reporter sequence is composed of DNA.

13. The method of claim 8, wherein said upstream oligonucleotide comprises a 3′

region configured to not hybridize to said stem region of said stem oligonucleotide.

14. The method of claim 8, wherein said upstream oligonucleotide comprises a 3′

region configured to hybridize to said stem region of said stem oligonucleotide.

15. The method of claim 8, wherein said stem specific region of said upstream oligonucleotide is between 5 and 12 bases in length.

16. A method for nucleic acid dispensing comprising;

a) dispensing a nucleic acid mixture comprising a plurality of oligonucleotides onto a first surface region such that a first amount of said oligonucleotides are delivered to said first surface region, wherein said dispensing is through a hydrophobic polymer dispensing component attached to a liquid dispensing device, and wherein said nucleic acid mixture further comprises a non-ionic detergent; and

b) dispensing said nucleic acid mixture into at least 15 additional surface regions through said hydrophobic polymer dispensing component attached to said liquid dispensing device such that the amount of said oligonucleotides delivered to each of said at least 15 additional surface regions is within about 15 percent of said first amount.

17. The method of claim 16, wherein said hydrophobic polymer dispensing component comprises a pipette tip.

18. The method of claim 16, wherein said hydrophobic polymer dispensing component is composed of a material comprising polypropylene.

19. The method of claim 16, wherein said amount of said oligonucleotides delivered to each of said 15 additional surface regions is within about 10 percent of said first amount.

20. The method of claim 16, wherein said amount of said oligonucleotides delivered to each of said 15 additional surface regions is within about 7 percent of said first amount.