Biotin Labelled Probes, Nucleic Acid Technology, Labelling Nucleic Acids, The System Exploits, Glycoprotein, Biotinylated

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Chimeric DNA Molecular Probes and Gene Libraries Chimeric DNA Molecular Probes and Gene Libraries Introduction Restriction Enzymes for Cloning Technique of Restriction Mapping Restriction Clevage and Gel Electrophoresis Construction of a Restriction Map Use of Partial Digests end Labelling and Hybridization in Restriction Mapping Construction of Chimeric DNA Cloning of Plasmid Vectors Directional Cloning of DNA Fragments with Heterologus Protruding Termini Adding Poly dA at 3' Ends of DNA Clone Blunt End Ligation by T4 DNA Ligase Cloning in Phage λ and Cosmid Vectors Cloning in phage λ Vectors Cloning in Cosmid Vectors Hosts for Cloning Vectors Bacteria as Hosts Eukaryotes as Hosts Molecular Probes Preparation of Probes Genomic DNA Probes

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Biotin labelled probes

Advances in nucleic acid technology later offered alternatives to radioactively labelled probes. One procedure that became increasingly popular in late 1980s is biotin labelling of nucleic acids. This system exploits the affinity, which the glycoprotein 'avidin' has for biotin (vitamin H). Avidin is commonly found in egg white.

Biotinylated probes are prepared through a nick-translation reaction by replacing nucleotides with biotinylated derivatives. After hybridization and washing, detection of hybrids is done by a series of cytochemical reactions which finally give a blue colour whose intensity is proportional to the amount of biotin in the hybrid.

There are several advantages of using biotinylated probes. For example, these assays employ non-toxic materials, whose half-life is longer. These probes can be prepared in advance in bulk and stored at -20.C for repeated uses. Detection of hybrids is much faster than in. case of radioactive probes. A limitation of this technology is that a very small probe (20 nucleotides) contains only a small number of biotinylated sites, limiting the intensity of signal obtained.

It has been solved by adding long 'tails' of biotinylated nucleotides to the probes through enzymatic methods. Sometime the probe does not need to be labelled with biotin, but only coupled with a tail. Another disadvantage of biotin labelled probes is that the cytochemical visualization reactions lead to precipitation of insoluble material which can not be removed and therefore, the filter can not be reused, while in radiolabelled probes, the filters can be repeatedly used for hybridization with a number of probes, one at a time.

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c DNA Probes Synthetic Oligonucleotides as Probes RNA Probes or Riboprobes Labelling of Probes Radiolablled Probes Non Radioactive Probes Biotin Labelled Probes Digoxigenin Labelled Probes Alternatives to Biotin and Digoxigenin Labelling Amplification of DNA Probe Signals Techniques Used in Molecular Probing Southern Nothern and Western Blotting Southern Blotting Northern Blotting Western Blotting Dot Blots and Slot Blots Applications of Molecular Probes Construction and Screening of Genomic and cDNA Libraries Genomic Library by Shotgun Experiment cDNA Library from mRNAs Colony or Plaque Hybridization for Screening of Libraries Chromosome Walking and Characterization of Chromosome Segments Chromosome Jumping or Hopping Libraries BAC Libraries and Assembly of BACs into Contigs