This chapter describes the methods used to isolate a cDNA for a protein closely related to the catalytic subunit of cAMP-dependent protein kinase. The same general technique could be used to isolate a cloned cDNA for any protein kinase or in fact for any protein for which a portion of the amino acid sequence was known. The general strategy for isolation of a cloned cDNA involves the use of oligonucleotides predicted from the amino acid sequence of the protein. The use of this technique is based on the development of hybridization and washing conditions which could discriminate against a single nucleotide mismatch between an oligonucleotide probe and a target nucleic acid. This allows identification of recombinant clones which contain a portion of the coding sequence of a particular protein. As the genetic code is redundant, it is necessary to synthesize a mixture of oligonucleotides containing all possible coding sequences. It is desirable to select a region of amino acid sequence which predicts a minimum number of coding sequences. Thus, regions containing methionine or tryptophan, which both have only a single codon, are desirable.
ASJC Scopus subject areas
- Molecular Biology