T-structure invasive cleavage assays, consistent nucleic acid dispensing, and low level target nucleic acid detection
First Claim
Patent Images
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 a 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, andiii) said polymerase extends a 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 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 oligonucleotides as templates such that stem amplicon sequences are generated.
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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.
77 Citations
11 Claims
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1. A method of target nucleic acid dependent amplification of a non-target sequence in a sample comprising;
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a) incubating a sample with stem oligonucleotides, upstream oligonucleotides, primers, dNTPs, and a polymerase under conditions such that, if a 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, andiii) said polymerase extends a 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 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 oligonucleotides as templates such that stem amplicon sequences are generated. - View Dependent Claims (2, 3, 4, 5)
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6. A method comprising:
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1) incubating a sample with stem oligonucleotides, upstream oligonucleotides, primers, dNTPs, and a polymerase under conditions such that, if a 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, andC) said polymerase extends a 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;2) heating said sample to separate said extended upstream oligonucleotides from said stem oligonucleotides and said target nucleic acid; and 3) 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. - View Dependent Claims (7, 8, 9, 10, 11)
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Specification