T-STRUCTURE INVASIVE CLEAVAGE ASSAYS, CONSISTENT NUCLEIC ACID DISPENSING, AND LOW LEVEL TARGET NUCLEIC ACID DETECTION

• US 20100285488A1
• Filed: 07/20/2010
• Published: 11/11/2010
• 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 8,354,232 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 of target nucleic acid dependent amplification of a non-target sequence in a sample comprising;

a) incubating a sample with stem oligonucleotides, upstream oligonucleotides, primers, dNTPs, and a polymerase under conditions such that, if the target nucleic acid is present;

i) a 3′

target specific region of said stem oligonucleotides hybridizes to said target nucleic acid, and a stem region of said stem oligonucleotides remains available for hybridization to said upstream oligonucleotides,

ii) a 5′

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

iii) said polymerase extends the 3′

end of said upstream oligonucleotides using said stem region of said stem oligonucleotides as a template to generate extended upstream oligonucleotides that comprise an upstream oligonucleotide extended region;

b) heating said sample in order to separate said extended upstream oligonucleotides from said stem oligonucleotides and said target nucleic acid;

c) cooling said sample under conditions such that said primers hybridize to at least a portion of said upstream oligonucleotide extended region of said extended upstream oligonucleotides; and

d) incubating said sample under conditions such that said primers are extended by said polymerase using said extended upstream oligonucleotide as a template such that stem amplicon sequences are generated.

2. The method of claim 1, further comprising the step of detecting the presence or absence of said target nucleic acid in said sample by determining if stem amplicon sequences are generated.

3. The method of claim 1, further comprising the step of performing one or more rounds of PCR using said stem amplicon sequences and said extended upstream oligonucleotides as templates, wherein said upstream oligonucleotides prime polymerization from said stem amplicon sequences, and wherein said primers prime polymerization from said extended upstream oligonucleotides.

4. The method of claim 3, further comprising a step of detecting the presence or absence of said target nucleic acid in sample sample by detecting any accumulated PCR products.

5. The method of claim 1, further comprising incubating said sample with downstream probes and upstream oligonucleotides such that invasive cleavage structure are formed with said stem amplicon sequences, the downstream probes, and the upstream oligonucleotides.

6. A method comprising:

incubating a sample with stem oligonucleotides, upstream oligonucleotides, dNTPs, and a polymerase under conditions such that, if the target nucleic acid is present;

A) a 3′

target specific region of said stem oligonucleotides hybridizes to said target nucleic acid, and a stem region of said stem oligonucleotides remains available for hybridization to said upstream oligonucleotides,

B) a 5′

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

C) said polymerase extends the 3′

end of said upstream oligonucleotides using said stem region of said stem oligonucleotides as a template to generate extended upstream oligonucleotides that comprise an upstream oligonucleotide extended region.

7. The method of claim 6, further comprising the step of heating said sample in order to separate said extended upstream oligonucleotides from said stem oligonucleotides and said target nucleic acid.

8. The method of claim 7, further comprising the step of cooling said sample under conditions such that said primers hybridize to at least a portion of said upstream oligonucleotide extended region of said extended upstream oligonucleotides.

9. The method of claim 8, further comprising the step of incubating said sample under conditions such that said primers are extended by said polymerase using said extended upstream oligonucleotide as a template such that stem amplicon sequences are generated.

10. The method of claim 9, further comprising the step of detecting the presence or absence of said target nucleic acid in said sample by determining if stem amplicon sequences are generated.

11. The method of claim 9, further comprising the step of performing one or more rounds of PCR using said stem amplicon sequences and said extended upstream oligonucleotides as templates, wherein said upstream oligonucleotides prime polymerization from said stem amplicon sequences, and wherein said primers prime polymerization from said extended upstream oligonucleotides.

12. The method of claim 11, further comprising a step of detecting the presence or absence of said target nucleic acid in said sample by detecting any accumulated PCR products.

13. The method of claim 9, further comprising incubating said sample with downstream probes and upstream oligonucleotides such that invasive cleavage structure are formed with said stem amplicon sequences, the downstream probes, and the upstream oligonucleotides.