3′ end tagged oligonucleotides
View Patent ↗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.
1. A method of producing a preparation of oligonucleotides having a reduced number of 5′ oligonucleotide fragments, comprising;
a) providing a solid support comprising a plurality of affinity groups;
b) synthesizing a plurality of oligonucleotides in the 3′ to 5′ direction such that the 3′ ends of said oligonucleotides are attached to said affinity groups;
c) while said 3′ ends of said plurality of oligonucleotides remain attached to said solid support via said affinity groups, treating said oligonucleotides with an agent that cleaves oligonucleotides at abasic sites, such that oligonucleotides containing abasic sites are cleaved, to produce a mixture of 5′ oligonucleotide fragments, 3′ oligonucleotide fragments, and uncleaved oligonucleotides;
d) separating said 5′ oligonucleotide fragments from said solid supports attached to 3′ oligonucleotides fragments and said uncleaved oligonucleotides;
e) cleaving said uncleaved oligonucleotides and 3′ oligonucleotide fragments from said solid support to generate a mixture comprising a plurality of released uncleaved oligonucleotides and 3′ oligonucleotide fragments comprising 3′ end affinity groups and lacking abasic sites; and
f) purifying said plurality of released uncleaved oligonucleotides and 3′ oligonucleotide fragments employing said 3′ end affinity groups to generate a preparation of oligonucleotides having a reduced number of 5′ oligonucleotide fragments.
2. The method of claim 1 , wherein said solid support comprises controlled pore glass.
3. The method of claim 1 , wherein said plurality of affinity groups comprise lipophilic moieties.
4. The method of claim 1 , wherein said affinity groups comprise long-chain polycarbon linkers.
5. The method of claim 4 , wherein said long-chain polycarbon linker comprises C 16 or C 14 .
6. The method of claim 1 , wherein said agent comprises aqueous lysine.