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Core Concepts
Scientific Fundamentals Explained

Which biotin modification to use?

Biotin in biotechnology 

Biotin is an important molecule in molecular biology applications, in part due to its very high affinity for streptavidin and avidin (see the prior DECODED 1.2 article, How Biotin Became a Tool of Molecular Biologists). It is used in mobility shift assays, and for enrichment, purification, and attachment to solid surfaces. Biotin can also be used for tagging target molecules with dye- or enzyme-labeled streptavidin. The affinity of the biotin–streptavidin interaction is extremely strong, with a disassociation constant (Kd) of 10−15 M. Biotin fits inside a ‘pocket’ (Figure 1) of the streptavidin protein [1], creating an enveloping effect which supple-ments the binding interaction. It is this binding strength that makes biotin a very useful tool for molecular and biomedical research.
Figure 1. Biotin–Streptavidin interaction. Biotin (green, red, and blue spheres) fits inside a pocket formed by the streptavidin protein (blue ribbon) [2].

Choosing a biotin modification 

Biotin can be added to oligonucleotides on either terminus (“standard” Biotin), as well as internally through a modified thymidine residue (Biotin-dT). There are several types of biotin modifications, each with their own benefits. However, it is important to note that each of the different biotin modifications contains the same functional biotin group. 

Standard Biotin. IDT offers a biotin modification that is attached to the 5’- or 3’-ends of an oligo using a C6 (standard) spacer (Figure 2). Referred to by IDT as “Biotin”, this version is recommended for most applications.

Figure 2. “Standard” Biotin. This version is used for addition to the 3’ end.

Figure 3. Biotin dT.

Biotin dT
. Biotin-modified thymidine residues (Figure 3) allow the addition of biotin internally within an oligonucleotide. Biotin dT residues can also be added to either end of an oligo.

Biotin-TEG. Biotin-TEG increases the oligo–biotin distance to 15 atoms using a triethyleneglycol (TEG) spacer. Biotin-TEG is commonly used to avoid hindrance issues and can be beneficial for attaching oligonucleotides to nanospheres or magnetic beads. 

Dual Biotin. Dual Biotin is a modification resulting in two functional biotin groups (Figure 4), which act to increase biotin–streptavidin binding affinity, and are used for applications requiring high sensitivity—e.g., Serial Analysis of Gene Expression (SAGE) assays.

Figure 4. Dual Biotin.

PC Biotin and DesthioBiotin-TEG. One of the most challenging, and often frustrating, aspects of applications employing biotin is the nearly irreversible biotin–streptavidin interaction. The bond is stable over a broad pH and temperature spectrum. Conditions necessary to release biotin can be potentially harmful or negatively affect downstream procedures. There are two biotin modifications that provide binding and controlled release:

PC Biotin (Figure 5) employs a photocleavable spacer arm which can be cleaved when exposed to UV light of specific wavelength (300–350 nm). One benefit of the PC modification is that upon cleavage, the resulting DNA oligo will have a free phosphate group available for subsequent ligase reactions.

Figure 5. Photocleavable (PC) Biotin.

, a biotin analog missing the sulfur atom, is another option for post-binding release. This analog binds tightly to streptavidin, but more weakly than standard biotin. Because of this, rinsing streptavidin bound oligos with buffered solutions containing free biotin will result in the displacement of desthiobiotin with the free biotin, allowing the oligo to be removed and collected [3].

Biotin Azide. Finally, biotin can be added to oligonucleotides either at the 5’ end or internally, using a reactive azide group in a click chemistry reaction (see the article, Oligo Modification—Post-Synthesis Conjugation Explained, in this issue, for more information about this reaction).

Ordering from IDT

Biotin modifications are available for ordering from IDT and can be found listed under the 5’, 3’, and Internal Modifications Tabs on the oligonucleotide ordering web page. Please note that most biotin modifications require a minimum processing scale of 100 nmoles and HPLC purification.

The IDT Modifications web page provides further structures and informa­tion about these modifications for biotinylation. For further questions regarding biotin options from IDT, please contact IDT Technical Support at custcare@idtdna.com.


  1. DeChancie J, Houk KN (2007) The origins of femtomolar protein–ligand binding: hydrogen bond cooperativity and desolvation energetics in the biotin–(strept)avidin binding site. J Am Chem Soc, 129(17):5419–5429.
  2. Weber PC, Ohlendorf DH, et al (1989) Structural origins of high-affinity biotin binding to streptavidin. Science, 243:85–88.
  3. Hirsh JD, Eslamizar l, et al. (2002) Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal Biochem, 308(2):343–357.

Product focus—Custom oligos and modifications

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.

Learn more or order now.

Oligo modifications

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 noncat@idtdna.com.

Additional reading

A biotin:streptavidin alternative for non-radioactive hybridization assays—Digoxigenin—Modification Highlight: Learn about Digoxigenin—a modification suitable for use as an alternative to biotin/streptavidin for non-radioactive hybridization applications.

HRP for sensitive hybridization probes—Modification Highlight: HRP can be directly conjugated to hybridization probes to increase signal amplification in CARD-FISH protocols. Also use this oligo modification in nonradioactive immunoassays, northern/Southern blot analysis, and other in situ hybridization applications.

Fluorescent dyes with no licensing restrictions—a growing portfolio—Need fluorescent dyes suitable for commercial and diagnostic applications? These have no patent licensing restrictions. Review this table of Freedom Dye alternatives for commonly used proprietary dyes.

Need a library of related DNA or RNA oligo sequences?—Build variability into your oligo sequences by incorporating Mixed Bases. We offer mixes of multiple base types as well as nonstandard and modified bases.

Review other DECODED Online newsletter articles on oligonucleotide handling, and on modified oligonucleotide applications. Or browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Author: Jeremy Pritchard, BA is a Technical Service Representative at IDT.

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