| Attachment Chemistry / Linkers Modifications |
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AcryditeTM |
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AcryditeTM is an attachment chemistry based on an acrylic phosphoramidite that can be added to oligonucleotides as a 5’-modification. Acrydite-modified oligonucleotides covalently react with thiol-modified surfaces or can be incorporated into polyacrylamide gels during polymerization. |
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5'
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Adenylation |
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T4 RNA Ligase uses ATP to adenylate the 5'-end of a single-strand nucleic acid sequence. This activated adenylated-oligo is then covalently connected (ligated) to the 3'-OH of a second single-stranded sequence. Adenylated oligonucleotides containing a pyrophosphate linkage are substrates for T4 RNA Ligase in the absence of ATP (11). IDT will custom adenylate an oligonucleotide for use with RNA-Ligase using the chemical adenylation method of Unrau and Bartel (12). T4 RNA Ligase will use an adenylated DNA linker with similar efficiency as an adenylated RNA linker and IDT recommends use of adenylated DNA oligos for this application. Note that IDT requires blocking the 3'-end of an adenylated oligo so it cannot circularize; use of either 3'-Spacer C3 /3SpC3/ or dideoxycytosine /3ddC/ is preferred.
References
1. England, T.E., Gumport, R.I. and Uhlenbeck, O.C. (1977) Dinucleoside pyrophosphate are substrates for T4-induced RNA ligase. Proc Natl Acad Sci U S A, 74, 4839-4842.
2. Unrau, P.J. and Bartel, D.P. (1998) RNA-catalysed nucleotide synthesis. Nature, 395, 260-263. |
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5'
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Cholesteryl-TEG |
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Cholesterol can be conjugated to oligonucleotides and can facilitate uptake into cells. It has been used as a transfection aid for antisense oligos and siRNAs, both in vitro and in vivo. Cholesterol is a very hydrophobic modification that is best purified using RP-HPLC. |
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3'
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Digoxigenin NHS Ester |
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Digoxigenin is a small hapten that can be conjugated to amino-modified oligos. Anti-digoxigenin antibodies allow capture or detection of a digoxigenin-labeled oligo and can be used in a variety of assay formats much like biotin/streptavidin. |
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5'
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3'
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I-Linker |
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I-LinkerTM, developed at IDT, is a proprietary covalent attachment chemistry for oligonucleotides. The modifier is attached to the 5'-end of the oligo. I-LinkerTM can be substituted for amino modifications in many applications. In addition, I-LinkerTM expands the range of reactive groups that can be used for conjugation, including aldehyde and ketone-modified ligands or surfaces. |
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5'
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A primary amino group can be used to attach a variety of modifiers (such as fluorescent dyes) to an oligonucleotide or used to attach an oligonucleotide to a solid surface. Amino modifiers can be positioned at the 5’-end with either a standard (C6) or longer (C12) spacer arm. Amino modifications can be positioned at the 3’-end. Internal amino modifications can be introduced using an amino-dT base. |
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Amino Modifier C6 |
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5'
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Amino Modifier C12 |
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5'
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Amino Modifier C6 dT |
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5'
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Internal
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3'
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Amino Modifier |
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3'
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Uni-LinkTM Amino Modifier |
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Uni-LinkTM is an amino-modifier phosphoramidite from Clontech that provides a free primary amine attached to the 5'-end of an oligo via a six carbon aliphatic spacer arm. It is functionally interchangeable with Amino Modifier C6. |
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5'
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Internal
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Biotin-modified oligos bind tightly to streptavidin. The streptavidin can be labeled with fluorescent dyes and enzymes or mediate attachment to a solid surface. A variety of molecular biology assays and purification methods employ biotin. Biotin can be added to the 5’- or 3’-ends of an oligo using either a C6 (standard) or TEG (tetra-ethyleneglycol, 15 atom) spacer arm. 5' Biotin-TEG requires purification. Internal biotin modification can be introduced using a biotin dT base, which also requires additional purification. |
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Biotin |
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5'
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3'
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Biotin dT |
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5'
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Internal
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3'
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Biotin-TEG |
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Biotin-TEG has an extended 15-atom spacer arm. |
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5'
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3'
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Dual Biotin |
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Two biotin groups are sequentially placed on the 5’-end, which increases the efficiency of streptavidin binding. Dual Biotin is commonly employed in SAGETM protocols. |
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5'
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PC Biotin |
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PC Biotin products a photocleavable spacer between the biotin group and the DNA bases. Optimal cleavage is obtained with exposure to long-wave UV light in the 300-350 nm spectral range. Cleavage releases the oligo with a 5'-phosphate group. |
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5'
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A thiol group can be used to attach an oligo to a variety of fluorescent and nonfluorescent moieties or surfaces. Oligos containing thiol modifiers are shipped in their oxidized (disulfide) form and require chemical reduction by dithiothreitol (DTT) or Tris (2-carboxyethyl) phosphine (TCEP) prior to use. |
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Thiol Modifier C3 S-S |
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3'
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Dithiol |
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Dithiol can be inserted into an oligonucleotide at the 5' position, the 3' position or internally. Each insertion results in 2 SH group available for coupling with ligands or surfaces (linkage to gold surfaces in one popular application). The DTPA modification can be inserted in series so that 2 or even 3 groups can be positioned adjacent to each other to increase efficiency of ligand/surface interactions. Note that serial insertions will decrease oligo yield due to lower efficiency of incorporation of the DTPA phosphoramidite. |
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5'
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Internal
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3'
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Thiol Modifier C6 S-S |
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5'
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