Biotin is a very commonly used label for oligonucleotides, thanks to its high affinity for streptavidin. This property makes it ideal for a wide range of experiments, and in this ‘Know Your Oligo Mod’ blog post we’ll explore some of the options available for biotin tags and their uses.
Streptavidin is a bacterial protein that avidly binds biotin, an essential vitamin for many organisms. With a dissociation constant about 10-15 M, this is one of the strongest non-covalent interactions between a protein and ligand.
Streptavidin is secreted by Streptomyces avidinii to sequester biotin and inhibit the growth of competing microbes in its environment. Now, scientists use this phenomenon as a dependable system for isolating, enriching and analysing targets of interest.
Hybridisation detection assays
Biotin is one of the most popular non-radioactive labels for hybridisation-based detection methods.1 In Northern and Southern blotting, the RNA or DNA sample is run on a gel and a biotinylated probe is added, which hybridises to its complementary sequence.
When streptavidin conjugated to a reporter enzyme like alkaline phosphatase is applied, it binds to the biotin probes. This can then be detected by adding dyes that produce a coloured band to reveal the presence of the target sequence.
The same process applies to in situ hybridisation studies with immobilised cells or tissue sections. This reveals where the biotin-tagged oligo is located in the sample and thus where the nucleic acid sequence of interest is expressed.1 An alternative approach, fluorescence in situ hybridisation (FISH), uses oligo probes that are directly labelled with fluorophores, which can be more specific.1
Affinity purification
Biotinylated oligos are also popular for hybridising to cellular RNA in affinity purification studies.
When the oligos are incubated with crude cell extract, they hybridise with complementary RNA strands. These can then be isolated by passing the mixture through streptavidin-coated beads in a column. The tagged oligos and any binding partners will tightly bind to the beads. After washing away the unbound molecules, the molecules that selectively bind to a particular oligo sequence will remain.
Techniques like Chromatin Isolation by RNA Purification (ChIRP) and Capture Hybridisation Analysis of RNA Targets (CHART) use biotin-tagged oligos for affinity capture of long noncoding RNA molecules that bind to chromatin.2-4 By cross-linking the RNA to chromatin, researchers can better understand the role of these key regulators in health and disease.
Figure 1.
A. The biotin-tagged oligos (blue) hybridise with the target sequence (green) and not with other RNA sequences (black). B. When the mixture is passed through beads conjugated to streptavidin (pink), the biotin group binds tightly. C. After washing away unbound RNA, just the target sequence and capture oligo remain, which can then be eluted for further study.
Biotinylated primers
Biotinylated primers can be used to end-label PCR amplicons. After the PCR, the biotin-tagged DNA products can be captured using streptavidin for further purification or manipulation. A biotinylated primer on just one side can be used to isolate a single strand of the DNA amplicons.
Figure 2. A biotin tag attached to the 3' end of an oligo via a three-carbon linker.
Researchers working with limited DNA samples developed an approach using biotin to recover the source DNA after creating next-generation sequencing libraries.5 The Biotinylated Amplicon Sequencing (BAmSeq) method uses tagged primers to create NGS libraries, allowing the original template to be separated afterwards by passing through streptavidin beads.
This means the template DNA can be used for further studies, such as qPCR or digital PCR. This approach could benefit clinical cancer testing, where tissue or liquid biopsy samples can be limited.
Diagnostic applications
The potential uses of biotin-tagged oligonucleotides vary widely. In addition to fundamental molecular biology research, biotin is also helping to create new diagnostic assays. For example, recent research has used biotin-tagged oligonucleotides to develop:
- a DNA aptamer for diagnosing TB,6
- a rapid, specific, and sensitive assay for two pig pathogens,7
- an attomolar-sensitive detection assay of miRNA biomarkers for cancer.8
Biotinylation is also commonly used to immobilise oligos on planar sensor surfaces for biosensor chips and microarrays.
For a DNA microarray, a probe with a biotin tag can be attached to a silica surface covered with bound streptavidin.9 These probes will capture the complementary DNA sequence, allowing detection.
DNA microarrays can play an important role in clinical diagnostics for some conditions where sequencing is not required or would be too expensive or slow.
Attaching biotin to oligonucleotides
LGC Biosearch Technologies offers a range of options for attaching a biotin tag to your custom oligos. The biotin is covalently attached to a spacer arm, which reduces steric hindrance and provides easy access for streptavidin.
The biotin can be attached directly to either the 5' or 3' terminus of an oligonucleotide. Alternatively, biotin can be conjugated to a modified thymidine, which can participate in hybridisation. This thymidine can be placed anywhere in the custom oligo, including internal positions.
More than one biotin tag can be included in your custom oligo to increase the strength of binding to streptavidin.10
Adding biotin during oligonucleotide solid-phase synthesis with phosphoramidite chemistry is the preferred and more efficient method, offering precise control. Enzymatic labelling using biotin ligase is typically less convenient and less commonly used for routine biotinylation of synthetic oligos.
It’s clear that biotin-tagged oligonucleotides have a wide range of purposes, helping to detect and extract your nucleic acids of interest. From fundamental laboratory research to developing highly sensitive cancer tests, biotin is a powerful modification to add to your oligo design.
Explore our range of oligo modifications and please contact us for other variations of biotin mods not listed online. If you are based in EMEA, please email us at lys.eu@lgcgroup.com or call +45 87 32 30 00. If you are based in the Americas or APAC, please email us at info@biosearchtech.com or call 1.415.883.8400 or 1.800.436.6631 (US & Canada only).
Know Your Oligo Mod series
- 2'-O-methoxyethyl (2'-MOE)
- Locked nucleic acids
- Amino linkers
- 5-methyl deoxycytidine
- 5-Nitroindole – a universal base oligo modification
- BHQ® (Black Hole Quencher®) non-fluorescent quenchers
- Thio C6 linker
- 3' Spacer C3
- Phosphorothioate bonds
- ATTO dyes
References
- Green MR and Sambrook J (2022) Preparation of Labeled DNA, RNA, and Oligonucleotide Probes. Cold Spring Harb Protoc doi: 10.1101/pdb.top100578
- Chu C, Quinn J and Chang HY (2012) Chromatin isolation by RNA purification (ChIRP). J Vis Exp (61):3912 doi: 10.3791/3912
- Simon MD et al. (2011) The genomic binding sites of a noncoding RNA. Proc Natl Acad Sci USA 108(51):20497-20502 doi: 10.1073/pnas.1113536108
- Simon MD and Machyna M (2019) Principles and Practices of Hybridization Capture Experiments to Study Long Noncoding RNAs That Act on Chromatin. Cold Spring Harb Perspect Biol 11(11):a032276 doi: 10.1101/cshperspect.a032276
- Cravero K et al. (2018) Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity. Practical Laboratory Medicine 12:e00108 doi: 10.1016/j.plabm.2018.e00108
- Hazra S et al. (2025) Development of DNA aptamers towards detection of tuberculosis biomarker Ag85B in a fluorescence-based sensing platform. Anal Chim Acta 1357:344029. doi: 10.1016/j.aca.2025.344029
- Wang H et al. (2025) Development and application of a dual LAMP-LFD assay for the simultaneous detection of Streptococcus suis and Glaesserella parasuis. Front Cell Infect Microbiol 15:1575365 doi: 10.3389/fcimb.2025.1575365
- Finocchiaro G et al. (2025) Large gold nanoparticle release assay for attomolar detection of miRNA related to myelodysplastic neoplasms. Talanta 293:128037 doi: 10.1016/j.talanta.2025.128037
- Nimse et al. (2014) Immobilization Techniques for Microarray: Challenges and Applications. Sensors 14(12):22208-22229 doi: 10.3390/s141222208
- Bardin et al. (1993) Comparison of 3' and 5' biotin labelled oligonucleotides for in situ hybridisation. Histochemistry 100(5):387-92 doi: 10.1007/BF00268937
