Dynamic flux nucleic acid sequence amplification
Provided herein are dynamic flux nucleic acid sequence amplification methods. The dynamic flux nucleic acid sequence amplification methods described herein are capable of amplifying nucleic acid sequences within a narrow temperature range. In some aspects, the disclosure provides for real-time dynamic flux nucleic acid sequence amplification methods.
1. A real-time dynamic flux method of nucleic acid sequence amplification, comprising:
a. combining a pair of forward and reverse oligonucleotide primers with a target nucleic acid sequence to be amplified; and
b. amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence within a 15° C. temperature range that is defined by the area contained within the overlap of an annealing curve (T A ) of the pair of oligonucleotide primers and the denaturation curve (T D ) of the target nucleic acid sequence,
wherein each forward and reverse oligonucleotide primer has a melting temperature (T m ) within 15° C. of the T m of the target nucleic acid sequence, and
wherein thermocycling comprises:
i. denaturing the target nucleic acid sequence; and
ii. annealing of the forward and reverse oligonucleotide primers; and
iii. extension of the target nucleic acid sequence by the forward and reverse oligonucleotide primers,
c. simultaneously detecting the amplified target nucleic acid sequence during said amplifying step.
2. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein detecting occurs by monitoring fluorescence.
3. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein detecting occurs by monitoring fluorescence of a fluorescent dye that intercalates with double-stranded DNA.
4. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein detecting occurs by monitoring fluorescence of a sequence-specific oligonucleotide probe labelled with a fluorescent reporter.
5. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 10° C. temperature range.
6. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 5° C. temperature range.
7. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 2.5° C. temperature range.
8. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein amplifying the target nucleic acid sequence by thermocycling the pair of forward and reverse oligonucleotide primers and the target nucleic acid sequence occurs within a 2.5° C. to 10° C. temperature range around the melting temperature of the target nucleic acid sequence.
9. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein one of the pair of oligonucleotide primers has a greater melting temperature range difference with the melting temperature of the target nucleic acid sequence than the second of the pair of oligonucleotide primers.
10. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein the melting temperature of one of the oligonucleotide primers is the same as the melting temperature of the target nucleic acid sequence.
11. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein the melting temperature of at least one of the oligonucleotide primers is within 10° C. of the melting temperature of the target nucleic acid sequence.
12. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein the melting temperature of at least one of the oligonucleotide primers is within 5° C. of the melting temperature of the target nucleic acid sequence.
13. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein the melting temperature of at least one of the oligonucleotide primers is within 2.5° C. of the melting temperature of the target nucleic acid sequence.
14. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , wherein each of the pair of oligonucleotide primers is present in an approximately equimolar concentration.
15. The real-time dynamic flux method of nucleic acid sequence amplification of claim 1 , further comprising: adjusting the pH of the amplification reaction to increase the specificity of selectively amplifying the target nucleic acid over other nucleic acids.