POLAR DYES
0 Assignments
0 Petitions
Accused Products
Abstract
The present invention relates to novel polar fluorescent and quenchers dyes, and minor groove binder with enhanced polarity. The present invention further relates to methods of preparing oligonucleotide probes labeled with polar arsonate dyes under the condition of automated synthesis and method of using such probes.
24 Citations
13 Claims
-
1. A compound of Formula (VIII):
- 2. An oligonucleotide conjugate comprising an oligonucleotide substituted with a dye of Formula (VIII):
-
7. A method of directly detecting a target nucleic acid, comprising:
-
providing a dye-labeled oligonucleotide probe comprising an oligonucleotide conjugated to one or more dye compounds of Formula (VIII), the oligonucleotide further optionally comprising an MGB optionally substituted with one or more polar arsonate groups, adding the dye-labeled oligonucleotide probe to a biological sample, wherein the probe is complementary or substantially complementary to the target nucleic acid; allowing the probe to hybridize with the target nucleic acid under an appropriate condition and for sufficient amount of time; and detecting fluorescence emitted from the dye.
-
-
8. A method of preparing an oligonucleotide in an automated synthesis, using a dye reagent of Formula (VIII), wherein Rx is a phosphoramidite group, the method comprising:
-
performing automated synthesis of oligonucleotide using phosphoramidite method to provide a dye-labeled oligonucleotide; deprotecting —
As(═
O)(OR6)(OR7) or —
As(OR6)2(OR7)2 of the dye-labeled oligonucleotide to provide a dye-labeled oligonucleotide having negatively charged polar group selected from —
AsO32−
or —
AsO3H−
; andisolating the dye-labeled oligonucleotide from the reaction medium. - View Dependent Claims (9, 10)
-
-
11. A method of detecting amplification of a target sequence in real life using a TaqMan assay, the method comprising the steps of:
-
providing a dye-labeled oligonucleotide probe comprising an oligonucleotide conjugated to a quencher dye of Formula (IV) and a fluorescent dye of Formula (VIII), each dye having one or more polar arsonate group, wherein the probe is in a conformation allowing the fluorescent dye to be quenched by the quencher dye; adding the dye-labeled oligonucleotide probe to a PCR reaction medium; running the PCR temperature cycles to allow the probe to hybridize with the target and get cleaved by Taq polymerase when the target is present; and reading the fluorescence signal from the cleaved dye in the real time, wherein the quencher dye of Formula (IV) is;
-
-
12. A method of detecting amplification of a target sequence in real life using a Molecular Beacon assay, the method comprising the steps of:
-
providing a dye-labeled oligonucleotide probe comprising an oligonucleotide conjugated to a quencher dye of Formula (IV) and a fluorescent dye of Formula (VIII), each dye having one or more polar arsonate group, wherein the probe is in a conformation allowing the fluorescent dye to be quenched by the quencher dye; adding the dye-labeled oligonucleotide probe to a PCR reaction medium; running the PCR temperature cycles to allow the probe to hybridize with the target thereby separating the fluorescent dye and the quencher dye when the target is present; and reading the fluorescence signal from the unquenched fluorescent dye in the real time, wherein the quencher dye of Formula (IV) is;
-
-
13. A method of detecting amplification of a target sequence in real life using a Scorpion Probe assay, the method comprising the steps of:
-
providing a dye-labeled oligonucleotide probe comprising a primer, an oligonucleotide conjugated to a quencher dye of Formula (IV) and a fluorescent dye of Formula (VIII), each dye having one or more polar arsonate group, wherein the probe is in a conformation allowing the fluorescent dye to be quenched by the quencher dye; adding the dye-labeled oligonucleotide probe to a PCR reaction medium; running the PCR temperature cycles to allow the primer to hybridize with a target DNA, thereby extending the probe on target DNA; heat denaturing the extended probe which causes the quencher dye to disassociate; cooling the extended probe to allow for an internal rearrangement which causes the fluorescent dye to fluorescence in a target specific manner; and reading the fluorescence signal from the fluorescent dye in the real time, wherein the quencher dye of Formula (IV) is
-
Specification