| One of the earliest methods for the detection of specific DNA or RNA sequences was through the incorporation of radioactively labeled nucleotides into nucleic acid sequences that were complementary to those of DNA or RNA species of interest (1-4). Following hybridization to the target sequence, the radioactive probes were quantitated by scintillation counting, autoradiography, or by phosphorimaging. While radioactive labeling is relatively straightforward, it does have its downside. The half-life of P32 is relatively short (14 days), so that fresh P32 labeled nucleotides must be ordered on a weekly basis and precautions must be taken to minimize the exposure of lab personnel to the hazards of radiation. Furthermore, the cost of storage and disposal of radioactively labeled material is not trivial.
For these reasons, alternative methods of detection have been devised. Whereas radiolabeling entails the incorporation of radioactive nucleotides or inorganic P32 into the polynucleotide chain, nonradioactive methods rely on the incorporation of chemical groups that are not normally found in nucleic acids. Following hybridization to its target sequence, the chemical group provides a means of detecting the probe by an appropriate indicator system. One of the earliest and still most widely used chemical labels is biotin (5-7), also known as vitamin H and is important biochemically in the transfer of carbon dioxide during carbohydrate and fat metabolism. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a reporter enzyme, such as alkaline phophatase or horseradish peroxidase. The introduction of a colorigenic, fluorogenic, or chemiluminescent substrate that upon hydrolysis by the enzyme produces color or emits visible or UV light at a specific wavelength provides a very sensitive method for detection. The most rapid and sensitive detection systems rely on chemiluminescent substrates. An example is the horseradish peroxidase-luminol detection system. HRP catalyzes the oxidation of luminol in the presence of hydrogen peroxide, generating a highly reactive endoperoxide that emits light a 425 nm during its decomposition to its ground state. Alkaline phosphatase detection systems that use dioxetane based chemiluminescent substrates are also available. The most sensitive chemiluminescent substrates in this category are CSPD and CDP-star (8,9). Cleavage of the phosphate ester of CDP-star by alkaline phosphatase radically reduces the thermal stability of the dioxetane ring, which then decomposes with emission of light at 463 nm.
Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.
Oligonucleotides can be labeled with biotin either at the 3’ or the 5’ ends or within the nucleotide sequence itself by the incorporation biotin labeled deoxythymidine. At Gene Link we offer several different types biotin labeling.
Biotin phosphoramidite
With this reagent biotin can be added to the 5’ end of an oligonucleotide. For increased sensitivity, this reagent allows for branching so that multiple biotin molecules can be introduced at the same site.
Biotin TEG phosphoramidite
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