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US 2020/0370107 A1Published application
Beacon-mediated exponential amplification reaction (bear) using a single enzyme and primer
AssigneeThe Governors of the University of Alberta
InventorsXiaochun Chris LE, Hongquan ZHANG, Ashley Meagan NEWBIGGING
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Abstract
There is described Beacon-mediated Exponential Amplification Reaction (BEAR) Using a Single Enzyme and Primer.
Claims20
A Beacon-mediated Exponential Amplification Reaction (BEAR) system, comprising;
- a polynucleotide beacon, comprising:
- a polynucleotide hairpin (HP), comprising:
- a first overhang region comprising a fluorophore quencher conjugated to the 5′end of said first overhang region, a first stem region positioned 3′ to the first overhang region, a loop region positioned 3′ to the first stem region, a second stem region positioned 3′ to the loop region, such second stem region complementary to the first stem region, and a second overhang region positioned 3′ to the second stem region;
- a fluorophore-conjugated polynucleotide (FS), comprising a fluorophore moiety conjugated to the 3′end of said polynucleotide, said fluorophore-conjugated polynucleotide is adapted to reversibly bind to said first overhang region of said polynucleotide hairpin (HP), and said first stem region and loop region of said polynucleotide hairpin (HP),
- a polynucleotide primer configured to reversibly bind to a primer binding region that overlaps with said second stem region and said second overhang region of said polynucleotide hairpin (HP) when a target polynucleotide in a sample reversibly binds to said loop region of said polynucleotide hairpin (HP), or when an unbound fluorophore-conjugated polynucleotide (FS) reversibly binds to said first stem region and said loop region of said polynucleotide hairpin (HP);
- and
- a polypeptide with DNA polymerase activity.
4 more elements
A method of detecting a target polynucleotide in a sample, comprising:
- contacting a sample with a Beacon-mediated Exponential Amplification Reaction (BEAR) system, said system comprising;
- a polynucleotide beacon, comprising:
- a polynucleotide hairpin (HP), comprising:
- a first overhang region comprising a fluorophore quencher conjugated to the 5′end of said first overhang region, a first stem region positioned 3′ to the first overhang region, a loop region positioned 3′ to the first stem region, a second stem region positioned 3′ to the loop region, such second stem region complementary to the first stem region, and a second overhang region positioned 3′ to the second stem region;
- a fluorophore-conjugated polynucleotide (FS), comprising a fluorophore moiety conjugated to the 3′end of said polynucleotide, said fluorophore-conjugated polynucleotide is adapted to reversibly bind to said first overhang region of said polynucleotide hairpin (HP), and said first stem region and loop region of said polynucleotide hairpin (HP),
- a polynucleotide primer configured to reversibly bind to a primer binding region that overlaps with said second stem region and said second overhang region of said polynucleotide hairpin (HP) when a target polynucleotide in a sample reversibly binds to said loop region of said polynucleotide hairpin (HP), or when an unbound fluorophore-conjugated polynucleotide (FS) reversibly binds to said first stem region and said loop region of said polynucleotide hairpin (HP);
- and
- a polypeptide with DNA polymerase activity.
5 more elements
A kit, comprising:
- a polynucleotide beacon, comprising:
- a polynucleotide beacon, comprising:
- a polynucleotide hairpin (HP), comprising:
- a first overhang region comprising a fluorophore quencher conjugated to the 5′end of said first overhang region, a first stem region positioned 3′ to the first overhang region, a loop region positioned 3′ to the first stem region, a second stem region positioned 3′ to the loop region, such second stem region complementary to the first stem region, and a second overhang region positioned 3′ to the second stem region;
- a fluorophore-conjugated polynucleotide (FS), comprising a fluorophore moiety conjugated to the 3′end of said polynucleotide, said fluorophore-conjugated polynucleotide is adapted to reversibly bind to said first overhang region of said polynucleotide hairpin (HP), and said first stem region and loop region of said polynucleotide hairpin (HP),
- a polynucleotide primer configured to reversibly bind to a primer binding region that overlaps with said second stem region and said second overhang region of said polynucleotide hairpin (HP) when a target polynucleotide in a sample reversibly binds to said loop region of said polynucleotide hairpin (HP), or when an unbound fluorophore-conjugated polynucleotide (FS) reversibly binds to said first stem region and said loop region of said polynucleotide hairpin (HP);
- and
- a polypeptide with DNA polymerase activity.
5 more elements
Dependencies are inferred from the claim text.