Nuclease-Free Real-Time Detection of Nucleic Acids
AssigneeRoche Molecular Systems, Inc.
InventorNicolas Newton
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Abstract
The invention is a method for amplification and detection of nucleic acids using primers and at least one hybridization probe labeled with a first fluorescent moiety and a second moiety, capable of changing the fluorescence of said first fluorescent moiety. The method comprises the steps of effecting denaturation of said target, formation of hybrids between said primers and probe and said target and detecting the change in fluorescence of said first fluorescent moiety, upon formation of said hybrids. Reaction mixtures and kits for practicing the method of the present invention are also disclosed.
Claims22
A method for amplification and detection of a target nucleic acid in a sample comprising the steps of:
- (a) contacting a sample, possibly comprising a target nucleic acid, with a template-dependent nucleic acid polymerase, substantially lacking 5′-3′ nuclease activity, at least two primers, at least partially complementary to separate portions of said target, and at least one probe, at least partially complementary to a portion of said target, other than the portions complementary to said primers;
- wherein
- said probe has a first fluorescent moiety and a second moiety, capable of changing the fluorescence of said first fluorescent moiety;
- (b) subjecting the mixture of step (a) to conditions sufficient to permit denaturation of said target;
- (c) subjecting the mixture of step (b) to conditions sufficient to permit said primers and probe to form hybrids with said target;
- and
- (d) detecting the change in fluorescence of said first fluorescent moiety, upon formation of said hybrids.
A reaction mixture for amplification and detection of a target nucleic acid, comprising template-dependent nucleic acid polymerase, substantially lacking the 5′-3′ nuclease activity, at least two primers, at least partially complementary to separate portions of said target, and at least one probe oligonucleotide, at least partially complementary to a portion of said target, other than the portions complementary to said primers;
- wherein said probe as a first fluorescent moiety and a second moiety, capable of changing the fluorescence of said first fluorescent moiety.
A kit for amplification and detection of a target nucleic acid, comprising:
- template-dependent nucleic acid polymerase, substantially lacking 5′-3′ nuclease activity;
- at least two primers, at least partially complementary to separate portions of said target;
- and
- at least one probe, at least partially complementary to a portion of said target, other than the portions complementary to said primers;
- wherein
- said probe has a first fluorescent moiety and a second moiety, capable of changing the fluorescence of said first fluorescent moiety.
Dependencies are inferred from the claim text.