Biotinylation Guidelines—Creating Biotinylated DNA

What is biotinylation?

Biotinylation is widely used to enable isolation, separation, concentration, and further downstream processing and analysis of biomolecules. Proper biotinylation of the target molecule is essential to ensure high binding efficiency to the Invitrogen Streptavidin-Coupled Dynabeads.

There are a variety of commercially available biotinylation reagents that target different functional groups like primary amines, sulfhydryls, carboxyls, carbohydrates, tyrosine and histidine side chains, and guanidine and cytosine bases. All biotin reagents should contain a spacer arm, at least 6 C-atoms in length, to reduce steric hindrance. Several biotin modifications with longer carbon arms are also available to reduce the steric hindrance, like biotin-TEG with 15 carbon arm spacer.

Biotinylation of proteins and peptides

There are many commercially available biotinylation kits that enable simple and efficient biotin labeling of antibodies, proteins, and peptides. Biotin with different spacer arm lengths reduce steric hindrances associated with streptavidin binding and allow for efficient capturing of biotinylated proteins. Biotinylation reagents are provided as either water soluble or insoluble. They are also provided with reactivity toward a wide variety of groups and can be coupled to either primary amine, secondary amine, sulfhydryl, carboxyl, or phosphate groups of proteins or peptides. Photoreactive biotin compounds that react non-specifically with proteins and other biomolecules upon photoactivation by UV light are also available.

You can therefore choose an optimal biotinylation reagent specific for your application without inactivating your target molecule.

The high stability and binding capacity of the DNA on Dynabeads allows binding of the target proteins with kinetics similar to that of DNA in free solution.

Biotinylation of oligonucleotides

Immobilize the biotin-labeled DNA/RNA target sequence onto the beads and incubate with the cell extract. You can then isolate the bound proteins using magnetic separation. You can elute off the protein for characterization or apply the whole solid-phase complex directly, e.g., to DNase footprinting studies.

A dual biotin with two biotin molecules in sequence can increase binding strength with streptavidin. This helps to keep biotinylated DNA/primers on the beads during heating at higher temperatures, something that is a challenge for many customers. It has been seen that dual biotin prevents or effectively reduces leakage of biotinylated DNA from beads during heating (9).

Purification options:
Free biotin in the sample will reduce the binding capacity of the beads by occupying the binding sites on the streptavidin and hence is important to be removed effectively. Biotinylated oligonucleotides should be purified from unbound biotin using one of the following options:

• Cartridge
• HPLC (4)
• PAGE

More information for oligo purification options

Other nucleic acid biotinylation techniques

1. End-labeling using PCR with a biotinylated primer (5, 6)
2. Enzymatic incorporation of a biotin dUTP label
3. A biotin dUTP label can be incorporated enzymatically into a double-stranded DNA fragment through end-labeling, by use of Klenow DNA polymerase enzyme, nick translation or mixed primer labeling (5, 6) RNA polymerases, including T7, T3, and SP6 RNA polymerases which can incorporate dUTP into RNA transcripts in an in vitro transcription reaction
4. Photobiotinylation

The photoactivatable biotin can be incorporated randomly in the DNA fragment double-stranded DNA and single-stranded DNA or RNA. Photoactivatable biotin is simply added to the sample and irradiated with UV light.

Purification of biotinylated PCR products

Using Dynabeads Streptavidin, purification of biotinylated PCR products is not necessary, and beads can be added directly to the PCR reaction to capture biotinylated PCR products. However, if the concentration of unused biotinylated primer is in excess, it is advisable to remove them before adding the beads to the PCR reaction. Excess biotinylated primer in the PCR reaction will reduce the binding capacity of the beads. Run the PCR with limiting concentrations of biotinylated primer, or remove free biotinylated primer using PCR clean-up kits.

More about PCR clean-up kits

Biotinylation using cleavable reagents

The streptavidin-biotin interaction is the strongest known non-covalent, biological interaction between a protein and ligand. The bond formation between biotin and streptavidin is very rapid and, once formed, is unaffected by wide extremes of pH, temperature, organic solvents and other denaturing agents. Hence, often very harsh methods are required to dissociate the biotin from streptavidin which will leave the streptavidin adversely denatured. Using derivative forms of biotin allow for a gentle way of dissociation of biotin from streptavidin. Several cleavable or reversible biotinylation reagents allow specific elution of the biotinylated molecule from streptavidin in a gentle way.

Biotinylation with cleavable reagents can be done in different ways:

1. Enzymatic incorporation of a biotin dUTP analogue with a cleavable linker. Incorporation of a biotin with a linker arm containing a disulphide bond allows for a simple dissociation of the DNA fragment, as the disulphide links easily become cleaved with dithiothreitol (DTT). This reagent is enzymatically incorporated into a DNA fragment either by end-labeling, nick translation, or mixed primer labeling.
2. Chemical incorporation of the guanido analogue of NHS biotin. 
3. Chemical biotinylation of proteins using a biotin-X-NHS-Ester (1). NHS-biotin contains a cleavable disulphide bond so the desired protein can be easily cleaved from the biotin/streptavidin complex (2). Thiol-cleavable NHS-activated biotins react efficiently with primary amine groups (-NH 2) in pH 7-9 buffers to form stable amide bonds.
4. DSB-XTM Biotin Protein Labeling Kit. This kit provides a method for efficiently labeling small amounts of antibodies or other proteins with the unique DSB-X biotin ligand. DSB-X biotin is a derivative of desthiobiotin, a stable biotin precursor that has the ability to bind biotin-binding proteins, such as streptavidin and avidin. Whereas harsh chaotropic agents and low pH (6.0 M guanidine HCl, pH 1.5) are required to dissociate the stable complexes formed between biotin and streptavidin or avidin, DSB-X biotin can be readily displaced by applying an excess of D-biotin or D-desthiobiotin at room temperature and neutral pH.

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8. Gibbs RA, et al (1990) Multiplex DNA deletion detection and exon sequencing of the hypoxanthine Phosphoribosyltransferase gene in Lesch-Nyhan families. Genomics 7:235–244.
9. Dressman D et al (2003) Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. PNAS 100(15):8817–8822.