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  • Methods of detection

    images32The specificity of molecular techniques is dependent on the stringency (“stiffness”) of the hybridization conditions, which is determined by a combination of different factors including temperature and salt concentration. The presence of a target sequence in a clinical specimen is demonstrated by the formation of a hybrid between the target nucleic acid and probe. The probe can be labeled directly or indirectly, a detectable molecule. The direct labeling can be done with radioisotopes (eg, P 32 or S 35) or fluorescent molecules (eg fluorescein or rhodamine). The probes can be labeled in position 5 ‘or 3’ through enzymatic reactions, it is also possible internal marking positions by incorporating, via polymerization of modified nucleotides. The indirect labeling depends on the use of a pair of affinity ligands such as biotin and streptavidin, thus, the probes can be labeled with biotin at one of the different possible positions, and after hybridization with the target sequence complex can react with a streptavidin-enzyme conjugate, to subsequently carry out the resulting complex detection by incubation with a suitable substrate for the enzyme.

    One shortcoming of these methods is the possibility of cross hybridization of probes with different sequences at the target chosen. This problem is especially relevant in solution hybridization techniques. Although the hybridization solution offers the advantage of rapid kinetics, it is necessary to separate before the detection phase, the probe-target hybrid probe unbound, which makes the automation of these techniques. To reduce this problem relies on methods such as Southern (DNA) and Northern (RNA) blotting, in which there is a separation of nucleic acid molecules by electrophoresis, to subsequently be transferred to a solid support. Immobilized nucleic acids are incubated under carefully controlled conditions in a solution containing the probe to ensure the highest level of specificity.


    Different systems have been described hybridization with two probes, the most typical format is one in which the two probes hybridize to different sequences of the same target, a probe is marked with a pickup group and the other with a detectable group. When the two probes react with the sample containing the target sequence occurs the formation of a complex capture probe-target-detection probe, subsequently be before the detection phase, the complex is separated from the probe not linked through the pickup group. The specificity of this system derives from the fact that they have produced two different hybridization events generating the complex capture probe-target-detection probe. However, it requires a careful control of hybridization conditions to maintain a high level of specificity.

    Published on November 4, 2012 · Filed under: Bioscience; Tagged as: , , , ,
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