Читать книгу Biomolecular Engineering Solutions for Renewable Specialty Chemicals - Группа авторов - Страница 39

Nucleic acid hybridization utilises short synthetic radiolabeled oligonucleotides called as probe. These probes are used for locating complementary sequences in individual cells or phages containing an insert. The success of the hybridization experiment relies upon the probe. Therefore, probe should be designed with care and should have some part complementary to the sequence of the cloned gene. If there is no information about the gene, then the sequences from the related protein can be derived and degenerate probes can be synthesized to be used. So, in this technique first the colonies are transferred to a nylon membrane and then lysis of cells will be done. The released DNA will be denatured through an alkali treatment. The denatured single‐stranded DNA will be heat treated so that they could bind to the membrane. After that the membrane will be submerged into solution containing DNA probes and incubated for a certain time to get hybridized by complementary base pairing. Finally, the hybridized radiolabeled probes will be identified by autoradiography.