Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2003-03-31
2004-06-15
Riley, Jezia (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S025300, C536S026600, C548S100000, C548S120000, C548S146000, C548S148000, C548S181000, C548S215000, C548S217000, C548S223000
Reexamination Certificate
active
06750024
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to dye compounds useful as quenchers in a reporter-quencher energy-transfer dye pair. More specifically, this invention relates to cyanine quencher compounds, reagents incorporating such compounds and methods utilizing such compounds and/or reagents.
BACKGROUND
Nucleic acid hybridization assays comprise an important class of techniques in modern biology. Such assays have diverse applications including the diagnosis of inherited disease, human identification, identification of microorganisms, paternity testing, virology, and DNA sequencing, i.e., sequencing by hybridization.
An important aspect of nucleic acid hybridization assays is the method used to facilitate detection of the hybridization event. A particularly important class of methods used in nucleic acid hybridization assays employs a reporter-quencher energy-transfer dye pair comprising a “reporter” dye and a “quencher” dye which interact through a fluorescence resonance energy transfer (FRET) process. In these methods, the reporter is a luminescent compound that can be excited either by chemical reaction, producing chemiluminescence, or by light absorption, producing fluorescence. The quencher can interact with the reporter to alter its light emission, usually resulting in the decreased emission efficiency of the reporter. This phenomenon is called quenching. The efficiency of quenching is a strong function of the distance between the reporter molecule and the quencher molecule. Thus, in a nucleic acid hybridization assay, detection of a hybridization event is accomplished by designing an energy transfer system in which the spacing between a reporter and a quencher is modulated as a result of the hybridization.
Quenchers which are presently used in FRET-based nucleic acid hybridization assays are themselves fluorescent. That is, in addition to quenching the fluorescence of the reporter, the quencher produces fluorescent emissions. This is problematic, particularly in assays employing multiple spectrally-resolvable reporters, because the quencher fluorescence can interfere with the fluorescent signal produced by one or more of the reporters.
Thus, there remains a continuing need for quencher dyes which are themselves substantially non-fluorescent.
SUMMARY
The present invention is directed towards our discovery of a class of non-fluorescent cyanine quencher compounds which are useful in the context of a reporter-quencher energy-transfer dye pair. These quencher compounds find particular application in nucleic acid hybridization assays employing fluorescence energy transfer as a means of detection.
In a first aspect, the invention comprises an asymmetric cyanine dye compound having the structure
including substituted forms thereof, wherein at least one of R
1
and R
2
is linking group, X is O, S, or Se, and n ranges from 0 to 2.
In a second aspect, the invention includes a reporter-quencher energy-transfer dye pair comprising a reporter dye and a quencher dye, wherein the quencher dye is an asymmetric cyanine dye compound of the first aspect.
In a third aspect, the invention includes a an oligonucleotide having a cyanine dye quencher according to the first aspect covalently attached thereto.
In a fourth aspect, the invention provides a method for detecting a target nucleic acid sequence including the steps of providing a sample nucleic acid including at least one target nucleic acid sequence, and hybridizing a labelled oligonucleotide probe to the target nucleic acid sequence, the labelled oligonucleotide probe being labelled with an asymmetric cyanine dye compound of the first aspect. In a particularly preferred embodiment of this fourth aspect, the method further includes the step of digesting the oligonucleotide probe such that one or both of the reporter and quencher dyes is removed from the oligonucleotide probe.
Various aspects and embodiments of the above-described invention achieve one or more of the following important advantages over known quencher dye compounds. The asymmetric cyanine quenchers of the present invention may be easily covalently linked to a reagent, e.g., a polynucleotide. Furthermore, oligonucleotide probes labelled with the asymmetric cyanine quenchers of the present invention exhibit enhanced hybridization stability as compared to conventionally labelled probes, thereby allowing for the use of shorter probes which are more sensitive to hybridization mismatches. In addition, the asymmetric cyanine quenchers of the present invention are essentially non-fluorescent, thereby providing additional spectrum which can be occupied by one or more additional reporters.
These and other objects, features, and advantages of the present invention will become better understood with reference to the following description, drawings, and appended claims.
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Graham Ronald J.
Haxo Francis T.
Lee Linda G.
Mullah Khairuzzaman B.
Andrus Alex
Applera Corporation
Grossman Paul
Liptak Vince
Riley Jezia
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