3' end tagged oligonucleotides

Abstract

The present invention provides compositions comprising oligonucleotides that have 3′ end groups (e.g. lipophilic moieties) that are useful in invasive cleavage reactions such as the INVADER assay. Specifically, the present invention provides compositions containing oligonucleotides with 3′ end groups configured for generating a detectable signal in invasive cleavages assays with a high signal-to-background ratio, as well as methods for generating such compositions.

We claim:  
     
         1 . A composition comprising a plurality of tagged oligonucleotides, wherein said tagged oligonucleotides comprise a lipophilic 3′ end group, and wherein said tagged oligonucleotides are configured to be cleaved by structure-specific enzymes in a first invasive cleavage reaction such that fragments are generated, wherein said fragments are configured to participate in a second invasive cleavage reaction in order to generate a detectable signal.  
     
     
         2 . The composition of  claim 1 , wherein said tagged oligonucleotides are configured to serve as probe oligonucleotides in said first invasive cleavage reaction.  
     
     
         3 . The composition of  claim 1 , wherein said fragments are configured to serve as INVADER oligonucleotides in said second invasive cleavage reaction.  
     
     
         4 . The composition of  claim 1 , wherein said lipophilic 3′ end group comprises a long-chain polycarbon linker.  
     
     
         5 . The composition of  claim 4 , wherein said long-chain polycarbon linker comprises C 16  or C 14 .  
     
     
         6 . The composition of  claim 1 , wherein said second invasive cleavage assay comprises FRET cassettes, and wherein said fragments are configured to hybridize to said FRET cassettes.  
     
     
         7 . A composition comprising: 
 a) a solid support,    b) a lipophilic moiety attached to said solid support, and    c) an oligonucleotide comprising a 3′ end and a 5′ end, wherein said 3′ end is attached to said lipophilic moiety, and wherein said oligonucleotide is configured to be cleaved by structure-specific enzymes in a first invasive cleavage reaction such that a fragment is generated, wherein said fragment is configured to participate in a second invasive cleavage reaction in order to generate a detectable signal.    
     
     
         8 . The composition of  claim 7 , wherein said oligonucleotide is configured to serve as a probe oligonucleotide in said first invasive cleavage reaction.  
     
     
         9 . The composition of  claim 7 , wherein said fragment is configured to serve as an INVADER oligonucleotide in said second invasive cleavage reaction.  
     
     
         10 . The composition of  claim 7 , wherein said second invasive cleavage assay comprises a FRET cassette, and wherein said fragment is configured to hybridize to said FRET cassette.  
     
     
         11 . The composition of  claim 7 , wherein said solid support comprises CPG.  
     
     
         12 . The composition of  claim 7 , wherein said lipophilic moiety comprises a long-chain polycarbon linker.  
     
     
         13 . The composition of  claim 12 , wherein said long-chain polycarbon linker comprises C 16  or C 14 .  
     
     
         14 . A method of synthesizing oligonucleotides, comprising; 
 a) providing a solid support comprising a plurality of affinity groups,    b) synthesizing a plurality of oligonucleotide in the 3′ to 5′ direction such that the 3′ ends of said oligonucleotides are attached to said affinity groups, wherein said oligonucleotides are configured to be cleaved by structure-specific enzymes in a first invasive cleavage reaction such that fragments are generated, wherein said fragments are configured to participate in a second invasive cleavage reaction in order to generate a detectable signal.    
     
     
         15 . The composition of  claim 14 , wherein said oligonucleotides are configured to serve as probe oligonucleotides in said first invasive cleavage reaction.  
     
     
         16 . The composition of  claim 14 , wherein said fragments are configured to serve as INVADER oligonucleotides in said second invasive cleavage reaction.  
     
     
         17 . The method of  claim 14 , further comprising c) treating said oligonucleotides with an agent such that abasic sites in said oligonucleotides are cleaved while said 3′ ends of said oligonucleotides remain attached to said solid support via said affinity groups.  
     
     
         18 . The method of  claim 14 , further comprising c) cleaving said oligonucleotides from said solid support to generate a plurality of cleaved oligonucleotides comprising 3′ end affinity groups.  
     
     
         19 . The method of  claim 18 , further comprising d) purifying said plurality of cleaved oligonucleotides employing said 3′ end affinity groups to generate a plurality of purified oligonucleotides.  
     
     
         20 . The method of  claim 14 , wherein said solid support comprises CPG.  
     
     
         21 . The method of  claim 14 , wherein said plurality of affinity groups comprise lipophilic moieties.  
     
     
         22 . The method of  claim 14 , wherein said affinity groups comprise long-chain polycarbon linkers.