Cloning Sequence-Specific DNA-Binding Factors from cDNA Expression Libraries Using Oligonucleotide Binding Site Probes
The method described in this chapter has been used in the molecular cloning of transcription factors and other factors with DNA-binding activity toward specific double-stranded DNA sequences. The protocol is based on the method of Singh et al. (1) and shares feature with the immunological approach to screening cDNA expression libraries (see Chapter 13). The principle is to probe a cDNA expression library (usually a λ-phage expression library) with a labeled double-stranded DNA probe containing the sequence recognized by the factor in question. Recombinants expressing a protein capable of binding the probe sequence in the presence of nonspecific competitor DNA are thus identified and can be isolated.
KeywordsMicrowave Agar Adenosine MgCl2 Polypeptide
- 2.Huynh, T. V., Young, R. W., and Davis, R. W. (1988) Construction and screening of cDNA libraries in lambda gt11 and lambda gt10, in Cloning: A Practical Approach, vol. 1 (Glover, D. M., ed.), IRL, Oxford, UK, pp. 49–78.Google Scholar