dUTP-based compositions for reducing primer-aggregate formations during nucleic acid amplification
Methods and compositions are provided for enhanced specificity and sensitivity of amplification reaction mixtures. Compositions in accordance with the present invention provide for reduced formation of primer-aggregates during amplification reactions. Reaction mixes include dNTPs, where a portion of the dNTPs has been replaced with an unconventional nucleotide, e.g., dUTP. Unconventional nucleotide concentrations are typically between 10% to 50% of the concentration of one of the standard dNTP. In some compositions the unconventional nucleotide is dUTP which replaces from about 10% to about 50% of the dTTP in the dNTP mix.
1. A reaction mixture for primer-based amplification and detection of a target nucleic acid, the reaction mixture comprising:
each conventional nucleotide dATP, dCTP, and dGTP, and a combination of unconjugated dUTP and dTTP in an amount equivalent to the concentrations of dATP, dCTP and dGTP, wherein said unconjugated dUTP replaces from about 10% to about 75% of said dTTP in said combination; and at least one of a fluorescent probe, beacon or intercalating dye;
wherein the inclusion of unconjugated dUTP reduces the formation of primer aggregates during the amplification reaction in comparison with an amplification reaction employing only conventional nucleotides; and
wherein said reaction mixture lacks a uracil degradation enzyme.
2. The reaction mixture according to claim 1 , wherein the unconjugated dUTP replaces from about 10 to about 30% of the dTTP in said combination.
3. The reaction mixture according to claim 1 , wherein the unconjugated dUTP replaces from about 20 to about 40% of the dTTP in said combination.
4. The reaction mixture according to claim 1 , further comprising at least one additional unconventional nucleotide, wherein the combined concentration of said unconjugated dUTP and said at least one unconventional nucleotide does not exceed 75% of any one conventional nucleotide in said reaction mixture.
5. The reaction mixture according to claim 1 , wherein said reaction mixture comprises a primer pair and wherein each member of the primer pair has at least one or more uracil bases incorporated therein.
6. The reaction mixture according to claim 5 , wherein each member of the primer pair has all of its thymidine bases replaced with uracil bases.
7. The reaction mixture according to claim 1 , wherein the unconjugated dUTP does not exceed a final amplification reaction concentration of about 300 μM.
8. The reaction mixture according to claim 1 , wherein the unconjugated dUTP does not exceed a final amplification reaction concentration of about 100 μM.
9. The reaction mixture according to claim 1 , further comprising at least one polymerase enzyme.
10. The reaction mixture according to claim 1 , further comprising a buffer system.
11. The reaction mixture according to claim 1 , wherein the reaction mixture further comprises sorbitol or mannitol.
12. The reaction mixture according to claim 11 , wherein the reaction mixture comprises 100 to 500 mM sorbitol or 100 to 200 mM mannitol.
13. A method for reducing primer aggregation during amplification and detection of target nucleic acid, the method comprising:
combining a target nucleic acid with a reaction mixture comprising each conventional nucleotide dATP, dCTP, and dGTP and a combination of dTTP with unconjugated dUTP in an amount equivalent to the concentrations of dATP, dCTP, and dGTP; wherein said unconjugated dUTP replaces from about 10% to about 75% of said dTTP in said combination;
amplifying the target nucleic acid to produce amplicons; and
detecting the amplicons so produced;
wherein the level of primer aggregate formed during the amplification step is reduced as compared to amplifying the target nucleic acid using a dNTP mix having only conventional nucleotides, wherein said method lacks an enzyme degradation step to degrade uracil-containing amplicons, and wherein said nucleic acid is DNA.
14. The method according to claim 13 , wherein the reaction mixture further comprises sorbitol or mannitol.
15. The method according to claim 14 , wherein the target nucleic acid has secondary structure.
16. The method according to claim 14 , wherein the reaction mixture comprises 100 to 500 mM sorbitol or 100 to 300 mM mannitol.
17. The method according to claim 13 , wherein the unconjugated dUTP replaces from about 10 to about 30% of the dUTP in said combination.
18. The method according to claim 13 , wherein the unconjugated dUTP replaces from about 20 to about 40% of the dTTP in said combination.
19. The method according to claim 13 , further comprising at least one additional unconventional nucleotide, wherein the combined concentration of said unconjugated dUTP and said at least one unconventional nucleotide does not exceed 75% of any one conventional nucleotide in said reaction mixture.
20. The method according to claim 13 , wherein the unconjugated dUTP does not exceed a final amplification reaction concentration of about 300 μm.
21. The method according to claim 13 , wherein the unconjugated dUTP does not exceed a final amplification concentration of about 100 μM.
22. The method according to claim 13 , further comprising at least one polymerase enzyme.
23. The method according to claim 13 , further comprising a buffer system.
24. The method according to claim 13 , wherein said amplifying step comprises heating said target nucleic acid to a temperature greater than 60° C.