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Biotin


Biotin plays an important role in many kinds of molecular biology applications, in part due to its very high affinity for streptavidin and avidin. It is employed in mobility shift assays, and enrichment, purification, and attachment to solid surfaces. Biotin can be also used for tagging target molecules with dye- or enzyme-labeled streptavidin. Creative Biolabs offers many types of biotin for oligonucleotide (oligo) modification.

Introduction

Biotin is an affinity label that can be incorporated at either the 5’- or 3’-end of an oligo, or at an internal position using biotin dT or amino bases for conjugation to biotin-NHS (N-hydroxysuccinimide ester (NHS ester) of biotin). Moreover, biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support in order to use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to the long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin.

Structure of Biotin. Figure 1. Structure of biotin. (Hirsch, 2002)

Why Uses Biotin?

Biotinylated oligos are most commonly employed as probes or primers in a variety of in vitro and in vivo applications. Besides as nucleic acid probes, biotinylated oligos are also useful for the purification of DNA binding proteins. The biotinylated oligo is able to be bound to a streptavidin matrix and used for either column or spin chromatography. In terms of the isolation of DNA binding proteins, the streptavidin-biotin-oligo complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the expected proteins that bind selectively to the oligo sequence can be eluted under conditions that disrupt the protein: DNA complex.

Types of Biotins

Biotinylated oligos have been used in a variety of biological assays and affinity purification applications. Creative Biolabs offers various types of biotins for oligo modification, listed below. It is noticeable that biotin is available to be added to the 5’- or 3’-end of an oligo using either a C6 (standard) or triethyleneglycol (TEG) spacer arm. At the same time, 5' biotin-TEG requires purification. Internal biotin modification can be introduced using a biotin dT base, which also requires additional purification.

Standard biotin is attached to the 5’- or 3’-end of an oligo using a C6 (standard) spacer.

Biotin-modified thymidine residues allow the addition of biotin internally within an oligo. What’s more, biotin dT residues can be added to either end of an oligo.

Biotin-TEG increases the oligo-biotin distance to 15 atoms using a TEG spacer. It is usually used to avoid hindrance issues and can be beneficial for attaching oligos to nanospheres or magnetic beads.

Dual biotin is a modification resulting in two functional biotin groups, which act to improve biotin-streptavidin binding affinity, and are employed for applications requiring high sensitivity (e.g., serial analysis of gene expression (SAGE) assays).

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

A biotin analog missing the sulfur atom, is another option for post-binding release. This analog binds tightly to streptavidin. Because of this, rinsing streptavidin bound oligos with buffered solutions containing free biotin will lead to the displacement of desthiobiotin with the free biotin, allowing the oligo to be removed and collected.

Biotin can be added to oligos either at the 5’ end or internally via using a reactive azide group in a click chemistry reaction.

Creative Biolabs offers several types of biotins to meet customers’ requirements. Furthermore, we provide high-quality products and technologies to effectively improve your projects. If you have any special requirements for biotin modified oligo modification services, please feel free to contact us.

Reference

  1. Hirsch, J.D.; et al. (2002). Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Analytical Biochemistry. 308(2), 343–357.

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