Many researchers are unaware that certain modifications may require the presence or addition of a specific nucleotide in their oligonucleotide sequence. These modifications come in many forms, including fluorophores, spacers, and attachment chemistry/linker modifications. The most common nucleotide that is introduced with these modifications is thymidine. Some of these modifications have “dT” included in their name, such as 3’ Biotin dT or Internal Fluorescein dT, while others are more difficult to identify. For example, Internal TAMRA NHS Ester (Figure 1) does not include dT in its name but is attached after oligonucleotide synthesis via an amino dT.
All internal NHS Esters and click-enabled modifications are attached through a thymidine residue after oligo synthesis. While most 3’ and 5’ modifications, including NHS Esters and click modifications, do not add an additional base to the oligo, 3’TAMRA in phosphoramidite form is one exception to this rule, as it is built from a dT residue.
Including a modification code (e.g., an internal TAMRA attachment) in your sequence during the ordering process will add a dT residue at that position. Thus, the modification code /i6-TAMN/ within the sequence TCGA/i6-TAMN/CGTA will incorporate a “T” nucleotide at that position. The final sequence will be: TCGA “T” CGTA, with TAMRA attached to the additional dT residue. It is therefore important for you to account for the necessary dT base in your sequence prior to ordering, i.e., when assessing binding to a target sequence and in any calculations, such as melting temperature (Tm).
Figure 1. Internal TAMRA NHS Ester. The structure shows the link between the TAMRA NHS Ester (red) and the amino dT (blue) required in the sequence for addition of this modification.
Review a list of the common modifications IDT can add to oligonucleotides here. Not finding a modification you need on the IDT website? No worries. IDT will consider any modification you need. Just send your request to firstname.lastname@example.org.
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Custom oligonucleotides and primers
You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on.
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Review a list of the common modifications IDT can add to oligonucleotides here. Not finding a modification you need on the IDT website? IDT will consider any modification you need. Just send your request to email@example.com.
Custom dsDNA fragments
Rather than annealing oligonucleotides to obtain dsDNA fragments, when your fragment size is 125 bp or longer, it might make more sense to order gBlocks® Gene Fragments. gBlocks Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.
Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.
Author: Michael Kammerer is the Manager of the Platinum HPLC Laboratories at IDT.
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