Marcatura delle sonde di DNA

Imagine an experiment where the goal is establishing the presence or absence of a specific gene in a bacterial genome. How might such a small nucleic acid sequence be detected within the complex genomic DNA? DNA probes can be used to achieve this – these are single-stranded DNA molecules that are complementary to the target nucleic acid sequence and labeled with a detection molecule. The most commonly used labels for DNA probes include radioisotopes like Phosphorus 32, fluorophores like fluorescein, or small molecule binding partners such as biotin and digoxigenin. These labels can be incorporated chemically after the synthesis of DNA probes or during their synthesis via one of the two methods. In the end-labeling method, the hydroxyl or phosphate of the nucleotide present at the 5’ or 3’ termini of the probe DNA sequence is covalently bound to the desired chemical group or label. For internal labeling, the nick translation method uses the DNase enzyme to make a small nick in one of the DNA strands followed by DNA polymerase I – which adds labeled and unlabeled nucleotides directly into the DNA strand. This results in a probe where one or more of the nucleotides is labeled – such as this one, labeled with biotin. Whichever the choice of labeling strategy, the double-stranded DNA probes need to be denatured into single-stranded DNA so that they can bind the target DNA. Additionally, the placement of the label on the DNA probe should be such that it does not interfere with the desired properties of the target sequence. The selection of the specific probe label is based on a variety of factors, including sensitivity, safety, probe stability, and method of detection. For example, radioisotopes are highly sensitive labels, and they can be directly detected through autoradiography. However, they have a relatively short shelf life and strict specific requirements for handling radiochemicals. Instead, small molecule binding partners like biotin may be used. Biotin is a water-soluble enzyme cofactor that can be detected based on its affinity for the streptavidin protein. When placed in solution together, biotin-streptavidin complexes form, meaning that the DNA probe is labeled with both molecules. The streptavidin protein itself is conjugated with a detection molecule, such as a fluorophore, which can be detected via fluorescence imaging at the corresponding wavelength. Alternatively, streptavidin can be labeled with reporter enzymes such as alkaline phosphatase or horseradish peroxidase. These probes can be detected upon the addition of a chromogenic or chemiluminescent substrate with the corresponding detection method. Although biotin-labeled probes are not as sensitive as radioisotopes, they have a longer shelf life and do not need special safety precautions for use.