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 defined by the melting temperature of the oligonucleotide primers and the melting temperature of the target nucleic acid sequence, and
wherein thermocy cling 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.