The Tetramethylammonium Chloride (TMAC) Method for Screening cDNA Libraries with Highly Degenerate Oligonucleotide Probes Obtained by Reverse Translation of Amino Acid Sequences

Abstract

If an unknown protem is purified and available in relatively small amounts, it is possible to determine the sequences of short internal peptides (1). In order to determine the whole sequence of the protein by cDNA cloning, one of the peptides of perhaps five to seven amino acids may be reverse translated into nucleotide sequence resulting in a 15–21 base long deoxyribonucleotide. Because of codon degeneracy, the number of possible oligonucleotides may be more than several hundred, which must be present in order to ensure that the correct sequence is represented. Only one of these oligonucleotides corresponds to the correct sequence. Traditionally long stretches of DNA are hybridized in buffered saline solution where physicochemical parameters affecting the annealing are well known (2, 3). However, for shorter DNA sequences, the melting temperature of each oligonucleotide depends on the G + C content, since G:C base pairs possessing three hydrogen bonds interact more strongly than A:T base pairs with two hydrogen bonds. The different oligonucleotides in a mixture will thus possess different melting temperatures. This means that in buffered saline solution, one usually chooses a melting temperature that is so low that the oligonucleotide with the lowest G + C content can hybridize. However, in doing so, it is possible that oligonucleotides with a higher G + C content may form stable hybrids with mismatches resulting in the cloning of artifact cDNAs. Even though this procedure has been used successfully (4-6), it is more convenient to use a different buffer type that contains tetramethylammonium chloride (TMAC), since it has been reported that this salt selectively binds to and stabilizes A:T base pairs so that their melting temperature becomes similar to that of G:C base pairs (7-9).