Detection of amplification products
View Patent ↗Compositions and methods are provided for quantitative detection of amplification products, the methods being suitable for multiplexing. A first oligonucleotide that includes a primer sequence for priming an amplification reaction and also is labeled with a fluorescent label or quencher is mixed with a second oligonucleotide which has a sequence suitable for hybridizing to a portion of the first oligonucleotide and has a fluorescent label if the first oligonucleotide has a quencher or a quencher if the first oligonucleotide has a fluorescent label; and a third nucleotide which includes some or all the primer sequence contained in the first oligonucleotide but is not labeled, the first and third oligonucleotide being combined in a molar ratio of 2.8 to 8.2.
1. A composition, comprising:
in a buffer,
(a) a first oligonucleotide for priming an amplification reaction, the first oligonucleotide comprising: i. a primer sequence that is complementary to a site in a target sequence, a tail sequence that is not complementary to the target sequence and that is 5′ of the primer sequence, and iii. a quencher or fluorescent label;
(b) a second oligonucleotide having a sequence that is hybridized to the tail sequence of the first oligonucleotide to form a duplex, the second oligonucleotide having a fluorescent label if the first oligonucleotide has a quencher label, or having a quencher label if the first oligonucleotide has a fluorescent label;
(c) a third oligonucleotide comprising some or all of the primer sequence contained in the first oligonucleotide and not including a quenching or fluorescent label, wherein the third oligonucleotide is capable of hybridizing with the site in the target sequence and with the second oligonucleotide; and
(d) a strand displacing polymerase.
2. A composition according to claim 1 , wherein the second oligonucleotide is at a concentration that is substantially the same as the first oligonucleotide.
3. A composition according to claim 1 , wherein the first oligonucleotide has a quencher label and the second oligonucleotide has a fluorescent label or the first oligonucleotide has a fluorescent label and the second oligonucleotide has a quencher label.
4. A composition according to claim 1 , wherein a molar ratio of the first oligonucleotide to the third oligonucleotide is in the range of 2:8 to 8:2.
5. A composition according to claim 1 , wherein the strand-displacing polymerases is Bst DNA polymerase, large fragment.
6. The composition according to claim 1 , wherein the first oligonucleotide and the third oligonucleotide are combined in at least 1:1 molar ratio.