FRET-based analytes detection and related methods and systems
FRET-based analytes detection and related methods and systems are described where a pair of FRET labeled primers and/or oligonucleotides are used that are specific for target sequences located at a distance up to four time the Förster distance of the FRET chromophores presented on the FRET labeled primers and/or oligonucleotides one with respect to the other in one or more polynucleotide analyte; in particular the pair of FRET labeled primers and/or oligonucleotides is combined with a sample and subjected to one or more polynucleotide amplification reactions before measuring FRET signals from at least one FRET chromophore.
1 . A kit to detect at least one polynucleotide analyte in a sample, the kit comprising at least one primer pair, wherein
each primer pair comprise:
a forward primer attached to a forward FRET chromophore,
a reverse primer attached to a reverse FRET chromophore,
wherein upon binding of the forward primer and the reverse primer to the polynucleotide analyte, the corresponding forward FRET chromophore and the reverse FRET chromophore are located within the Forster distance of the forward FRET chromophore and the reverse FRET chromophore or at a distance of about 1×, about 2×, about 3×, or about 4× the Forster distance of the forward FRET chromophore and the reverse FRET chromophore.
2 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance within the Forster distance of the first FRET chromophore and the second FRET chromophore.
3 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance of about 1× the Forster distance of the forward FRET chromophore and the reverse FRET chromophore.
4 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance of about 2× the Forster distance of the forward FRET chromophore and the reverse FRET chromophore.
5 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance of about 3× the Forster distance of the forward FRET chromophore and the reverse FRET chromophore.
6 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance of about 4× the Forster distance of the forward FRET chromophore and the reverse FRET chromophore.
7 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance of about 0.25 nm to about 30 nm.
8 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance of about 0.25 nm to about 28 nm.
9 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance of about 0.25 nm to about 20 nm.
10 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance of about 0.25 nm to about 15 nm.
11 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance of about 0.25 nm to about 10 nm.
12 . The kit of claim 1 , wherein the corresponding first FRET chromophore and the second FRET chromophore are located at a distance of about 0.25 nm to about 5 nm.