Biotin Labelled DNA

What is the biotin labelled DNA?

Biotin-labeled DNA denotes deoxyribonucleic acid molecules that have been chemically altered with biotin tags, a very flexible and extensively utilized alteration in molecular biology. Biotin, referred to as Vitamin B7 or Vitamin H, is a diminutive, hydrophilic molecule that exhibits remarkable affinity and specificity for avidin or streptavidin proteins, rendering it an ideal tag for detection, immobilization, and purification purposes. The core idea of biotin-labeled DNA is based on the biotin-streptavidin contact, recognized as one of the most robust non-covalent interactions in biological systems, characterized by a dissociation constant (Kd) about 10^-15 M.

Due to the robustness and specificity of the biotin-streptavidin combination, biotin-labeled DNA is utilized in several sophisticated biological applications, such as DNA hybridization tests, pull-down experiments, protein-DNA interaction investigations, and targeted gene therapy. Recent work emphasizes the adaptability of this labeling method, showcasing its application in next-generation sequencing, biosensing technologies, and chromatin immunoprecipitation (ChIP). The biotin label can be integrated into DNA molecules using enzymatic, chemical, or synthetic techniques. This encompasses the direct integration of biotinylated nucleotides during PCR (Polymerase Chain Reaction) or in vitro transcription, together with post-synthetic alterations of DNA termini with biotin-labeled phosphoramidites. The labeling procedure does not disrupt the inherent hybridization characteristics of DNA, enabling the identification and manipulation of biotinylated nucleic acids without impairing their biological activities.

The amplification product labelling and subsequent detection. (Warmt C., et al., 2024)

Graphical representation of flexible tandem biotin labeling. (Cai W., et al., 2023)

Applications of biotin-labeled DNA

Biotin-labeled DNA technology, due to its simplicity, accuracy, and robust affinity for streptavidin, has proven essential in several fields of molecular biology, biotechnology, and genomics. Recent research has broadened the use of this technique in high-throughput and very sensitive biological systems, where little background interference is essential.

DNA hybridization probes: Biotin-labeled DNA probes are commonly employed in both Southern and Northern blotting to identify particular nucleic acid sequences in complicated biological samples. The probes hybridize with their corresponding target sequences, and the biotin moiety is then identified using streptavidin coupled to reporter molecules, such as alkaline phosphatase or horseradish peroxidase (HRP). This enables precise and accurate identification of DNA or RNA across various blotting and hybridization methodologies. Biotin-labeled probes are essential in fluorescence in situ hybridization (FISH), allowing the observation of chromosomal aberrations and gene expression patterns with great spatial resolution.

Diagram of the ssDNA generation from biotinylated PCR product using streptavidin. (Marimuthu C., et al., 2012)

DNA purification and isolation: The biotin-streptavidin interaction is utilized in the purification of DNA molecules. Biotin-labeled DNA can be affixed to streptavidin-coated magnetic beads or columns, facilitating the targeted extraction of the labeled nucleic acid from intricate biological mixtures. This method is especially advantageous in affinity chromatography and pull-down tests, aimed at purifying specific DNA sequences, transcription factors, or protein-DNA complexes. Recent research has employed biotin-labeled DNA to investigate epigenetic changes and protein-DNA interactions that govern gene expression, highlighting its effectiveness in clarifying molecular pathways.

DNA-protein interaction studies: Biotin-labeled DNA has become an essential instrument for investigating DNA-protein interactions. Techniques like chromatin immunoprecipitation (ChIP) utilize biotinylated DNA fragments to isolate and examine DNA-associated proteins, including as histones and transcription factors, to elucidate gene regulation networks. In electrophoretic mobility shift assays (EMSA), biotin-labeled DNA facilitates the detection and characterisation of DNA-binding proteins by altering the mobility of protein-DNA complexes on polyacrylamide gels, with streptavidin-HRP acting as a sensitive detection method.

Next-generation sequencing and microarrays: In next-generation sequencing (NGS) and DNA microarray technologies, biotin-labeled DNA is essential for the identification and quantification of target DNA sequences. Biotin-labeled primers are employed to target particular DNA fragments during sequencing library preparation, facilitating their selective capture or enrichment. In DNA microarrays, biotin-labeled probes hybridize with target DNA sequences on the array surface, and the signal is enhanced using streptavidin-conjugated fluorophores or enzymes.

DNA breaks are end-labeled by the incorporation of biotin-dNTP at 3′OH termini. (Leduc F., et al., 2011)

Gene therapy and targeted delivery: Biotin-labeled DNA enhances the targeting efficacy of gene delivery methods. Biotinylated plasmids or oligonucleotides can be attached to streptavidin-modified carriers, including nanoparticles or viral vectors, facilitating targeted delivery to specific cells. Recent research in therapeutic gene editing has investigated the application of biotin-labeled CRISPR/Cas9 components to enable site-specific delivery and improve the efficacy of gene repair processes.

Preparation of biotin-labeled DNA

PCR incorporation: A prevalent method for generating biotin-labeled DNA is the incorporation of biotinylated nucleotides (e.g., biotin-dUTP) during PCR amplification. This approach allows for the production of tagged DNA fragments with biotin at various locations, thereby enhancing their detection and capture. The integration method is exceptionally efficient and compatible with many PCR techniques, rendering it suitable for applications including probe creation and molecular cloning.

End labeling with biotin: Biotin can be enzymatically conjugated to the 5' or 3' ends of DNA strands utilizing enzymes like T4 DNA polymerase or T4 polynucleotide kinase. This approach involves the addition of biotinylated nucleotides to the termini of DNA molecules in a highly selective reaction, enabling precise control over the labeling process. This method is very advantageous for annotating specific DNA sections or producing end-labeled oligonucleotides for hybridization research.

Chemical labeling: When enzymatic procedures are impractical, biotin can be chemically conjugated to DNA. Biotin-NHS (N-hydroxysuccinimide) esters can react with primary amines on modified DNA bases, resulting in a persistent covalent connection. This approach is exceptionally versatile, permitting the conjugation of biotin to both the backbone and the termini of DNA molecules.

In Vitro transcription: In RNA-based applications, biotin may be integrated into RNA transcripts by using biotin-labeled ribonucleotides (e.g., biotin-UTP) in in vitro transcription operations. This technique is frequently employed to produce biotin-labeled RNA probes for hybridization experiments and ribonucleoprotein investigations.

The biotinylated primers were similar to with the addition of biotin at the 5' end. (Cravero K., et al., 2018)

DNA conjugation technology at BOC Sciences

Advantages of biotin labelled DNA

High Affinity and Specificity: A primary benefit of biotin-labeled DNA is the exceptionally strong and specific interaction between biotin and streptavidin (or avidin). This non-covalent contact possesses a dissociation constant (Kd) of around 10^-15 M, rendering it one of the most robust biological connections. The elevated specificity of this interaction guarantees that the biotinylated DNA can be efficiently and dependably detected, captured, or immobilized in diverse experimental configurations with minimal background interference, which is advantageous in methodologies such as Southern blotting, chromatin immunoprecipitation (ChIP), and pull-down assays.

Versatile detection and visualization: Biotin-labeled DNA can be identified via various streptavidin-conjugated reporter molecules, such as enzymes (e.g., alkaline phosphatase, horseradish peroxidase), fluorophores, or quantum dots. This adaptability enables researchers to select the most suitable detection method for their respective applications, whether it entails colorimetric detection in blotting methods or fluorescence-based imaging in fluorescence in situ hybridization (FISH) or confocal microscopy. This versatility significantly enhances the use of biotin-labeled DNA in both low- and high-resolution detection techniques.

Non-invasive tagging: The biotin label is diminutive and hydrophilic, indicating that it generally does not disrupt the hybridization characteristics of DNA or its interactions with other molecules, including proteins. This enables biotin-labeled DNA to maintain its typical biological activities, rendering it appropriate for investigating DNA-protein interactions, hybridization of genetic material, and gene expression. The biotin tag's non-invasive characteristic guarantees the preservation of DNA's original structure, therefore averting detrimental alterations in DNA activity.

Disadvantages of biotin labelled DNA

Steric hindrance: The substantial biotin-streptavidin interaction can induce steric hindrance, potentially disrupting the inherent binding characteristics of the DNA. This may influence the interaction between DNA and its binding partners, including proteins, enzymes, or complementary DNA/RNA sequences.

Alteration of DNA Functionality: The incorporation of biotin may possibly alter the native structure or function of DNA, particularly if it is affixed near a functionally significant region (e.g., promoter sequences, protein recognition sites, etc.). Alterations at critical loci can hinder DNA's capacity to replicate, transcribe, or associate with proteins (such as transcription factors), hence restricting the application of biotin-labeled DNA in specific biological tests.

Limited Reversibility: The biotin-streptavidin connection is exceptionally robust, characterized by a dissociation constant in the femtomolar range, rendering it challenging to reverse under typical conditions. Releasing DNA trapped by streptavidin is difficult without denaturing either the DNA or the associated proteins. This restricts the capacity to retrieve functional, undamaged DNA for further application in future experiments.

Sensitivity to Environmental Conditions: The efficacy of the biotin-streptavidin interaction can be affected by specific environmental factors, such elevated salt concentrations, alterations in pH, or the presence of detergents. Under suboptimal conditions, the interaction between biotin and streptavidin may diminish, hence decreasing the sensitivity of assays dependent on biotin-labeled DNA. This may also complicate experimental optimization and repeatability.