(76)
7. Beskrive hvordan et specifik gen kan identificeres i et bibliotek (fx. genindentificering ved hybridisering af speciffike prober, hvordan prober mærkes til screening)
Devlin, s.296-7
Devlin, s.44

Selection of bacteria that harbour recombinant DNA of interest in a gene library requires sensitive and specific detection methods. DNA and RNA probes meet these requirements.

Probes – short-single stranded DNA or RNA oligonucleotides that are complementary to target nucleic acid and will hybridise with it. The degree of complementarity of a probe with the target DNA determines the tightness of binding to the probe. The probe does not need to contain the entire complementary sequence of the DNA.

Appropriate labels include radioactive elements, fluorescent chromophores and biotin.

1. The probes can be labelled by introducing random nicks (breaks in the phosphodiester bond of the backbone) by DNase I. Another enzyme, DNA polymerase I removes the bases in the nicks, creating gaps and filling them afterwards with radioactively marked nucleotides.
    

2. Another method produces longer labelled DNA probes. The double-stranded DNA is melted and hybridized with a mixture of random hexanucleases containing all possible nucleotides. They serve as primers for DNA synthesis in the presence of DNA polymerase and radioactive labelled nucleotides.
    

RNA molecules can also act as probes. They need not be melted prior to hybridisation. Synthesis of RNA however requires a primer and that is taken care of by specially constructed vectors.  

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