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US 10,240,178 B2Granted
Mispriming prevention reagents
AssigneeBrandeis University
InventorsLawrence J. Wangh, Kenneth E. Pierce, Jesus A. Sanchez, John E. Rice, Alexandra Isabel King Over, Adam E. Osborne
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Abstract
Provided herein are mispriming prevention reagents, compositions and kits comprising such reagents and methods of use thereof.
Claims24
A mispriming prevention reagent comprising a nucleic acid molecule comprising, in 5′ to 3′ order:
- (i) a first condition-dependent stem region comprising a 5′ terminal covalently linked moiety and a first stem nucleic acid sequence, wherein the first stem nucleic acid sequence is at least 6 nucleotides in length and wherein the 5′ terminal covalently linked moiety comprises a cyclic or polycyclic planar moiety that does not have a bulky portion;
- (ii) a condition-dependent loop region comprising a loop nucleic acid sequence of at least 3 nucleotides in length;
- and
- (iii) a second condition-dependent stem region comprising a second stem nucleic acid sequence and a 3′ terminal covalently linked moiety, wherein the second stem nucleic acid sequence is at least 6 nucleotides in length and is complementary to the first stem nucleic acid sequence and wherein the 3′ terminal covalently linked moiety comprises a cyclic or polycyclic planar moiety that does not have a bulky portion, wherein the 3′ terminal covalently linked moiety is non-identical to the 5′ terminal covalently linked moiety, and wherein the 3′ terminus of the second stem region is non-extendable by a DNA polymerase,
- wherein the first condition-dependent stem region hybridizes to the second condition-dependent stem region in a temperature dependent manner to acquire a stem-loop hairpin conformation, wherein the mispriming prevention reagent acquires a principally stem-loop conformation at temperatures below primer annealing temperature of an amplification reaction.
2 more elements
A reaction mixture comprising:
- (a) a first nucleic acid primer that hybridizes to a 3′ region of a target nucleic acid sequence with a first primer melting temperature;
- (b) a second nucleic acid primer that hybridizes to a 3′ region of the complement of the target nucleic acid sequence with a second primer melting temperature;
- and
- (c) a mispriming prevention reagent, wherein the mispriming prevention reagent comprises a nucleic acid molecule comprising, in 5′ to 3′ order:
- (i) a first condition-dependent stem region comprising a 5′ terminal covalently linked moiety and a first stem nucleic acid sequence, wherein the first stem nucleic acid sequence is at least 6 nucleotides in length and wherein the 5′ terminal covalently linked moiety comprises a cyclic or polycyclic planar moiety that does not have a bulky portion;
- (ii) a condition-dependent loop region comprising a loop nucleic acid sequence of at least 3 nucleotides in length;
- and
- (iii) a second condition-dependent stem region comprising a second stem nucleic acid sequence and a 3′ terminal covalently linked moiety, wherein the second stem nucleic acid sequence is at least 6 nucleotides in length and is complementary to the first stem nucleic acid sequence, wherein the 3′ terminal covalently linked moiety comprises a cyclic or polycyclic planar moiety that does not have a bulky portion, wherein the 3′ terminal covalently linked moiety is non-identical to the 5′ terminal covalently linked moiety, and wherein the 3′ terminus of the second condition-dependent stem region is non-extendable by a DNA polymerase, wherein the second stem region hybridizes to the first condition-dependent stem region with a stem melting temperature that is no greater than both the first primer melting temperature and the second primer melting temperature, and wherein hybridization of the first condition-dependent stem region to the second condition-dependent stem region causes the reagent to acquire a stem-loop hairpin conformation, wherein the mispriming prevention reagent acquires a principally stem-loop conformation at temperatures below primer annealing temperature(s) of an amplification reaction.
5 more elements
A method of creating an amplification product comprising a target nucleic acid sequence or complement thereof, the method comprising:
- (a) forming a reaction mixture comprising:
- (i) a target nucleic acid molecule comprising the target nucleic acid sequence;
- (ii) a first nucleic acid primer that hybridizes to a 3′ region of the target nucleic acid sequence with a first primer melting temperature;
- (iii) a second nucleic acid primer that hybridizes to a 3′ region of the complement of the target nucleic acid sequence with a second primer melting temperature;
- (iv) a thermostable DNA polymerase;
- (v) dNTPs;
- and
- (vi) a mispriming prevention reagent comprising a nucleic acid molecule comprising, in 5′ to 3′ order:
- (1) a first condition-dependent stem region comprising a 5′ terminal covalently linked moiety and a first stem nucleic acid sequence, wherein the first stem nucleic acid sequence is at least 6 nucleotides in length and wherein the 5′ terminal covalently linked moiety comprises a cyclic or polycyclic planar moiety that does not have a bulky portion;
- (2) a condition-dependent loop region comprising a loop nucleic acid sequence of at least 3 nucleotides in length;
- and
- (3) a second condition-dependent stem region comprising a second stem nucleic acid sequence and a 3′ terminal covalently linked moiety, wherein the second stem nucleic acid sequence is at least 6 nucleotides in length and is complementary to the first stem nucleic acid sequence, wherein the 3′ terminal covalently linked moiety comprises a cyclic or polycyclic planar moiety that does not have a bulky portion, wherein the 3′ terminal covalently linked moiety is non-identical to the 5′ terminal covalently linked moiety, wherein the 3′ terminus of the second condition-dependent stem region is non-extendable by the thermostable DNA polymerase, wherein the second condition-dependent stem region hybridizes to the first condition-dependent stem region with a stem melting temperature that is no greater than the first primer melting temperature and the second primer melting temperature, and wherein hybridization of the second condition-dependent stem region to the first condition-dependent stem region causes the reagent to acquire a stem-loop hairpin conformation,
- wherein the mispriming prevention reagent acquires a principally stem-loop conformation at temperatures below primer annealing temperature(s) of an amplification reaction;
- and
- (b) incubating the reaction mixture under conditions such that the first primer or second primer is extended by the thermostable DNA polymerase to create an amplification product comprising the target nucleic acid sequence or complement thereof.
12 more elements
Dependencies are inferred from the claim text.