The discordance between the number of nucleic acid bases and the number of amino acids immediately eliminates the possibility of a code of one base per amino acid. In fact, even two nucleotides per amino acid (a doublet code) could not account for 20 amino acids (with four bases and a doublet code, there would only be 16 possible combinations [42 = 16]). Thus, the smallest combination of four bases that could encode all 20 amino acids would be atriplet code. However, a triplet code produces 64 (43 = 64) possible combinations, or codons. Thus, a triplet code introduces the problem of there being more than three times the number of codons than amino acids. Either these "extra" codons produce redundancy, with multiple codons encoding the same amino acid, or there must instead be numerous dead-end codons that are not linked to any amino acid.
Preliminary evidence indicating that the genetic code was indeed a triplet code came from an experiment by Francis Crick and Sydney Brenner (1961). This experiment examined the effect of frameshift mutations on <span>protein synthesis</span>. Frameshift mutations are much more disruptive to the genetic code than simple base substitutions, because they involve a base insertion or deletion, thus changing the number of bases and their positions in a gene. For example, the mutagen proflavine causes frameshift mutations by inserting itself between DNA bases. The presence of proflavine in a DNA molecule thus interferes with the molecule's replication such that the resultant DNA copy has a base inserted or deleted.
Crick and Brenner showed that proflavine-mutated bacteriophages (viruses that infect bacteria) with single-base insertion or deletion mutations did not produce functional copies of the protein encoded by the mutated gene. The production of defective proteins under these circumstances can be attributed to misdirected translation. Mutant proteins with two- or four-nucleotide insertions or deletions were also nonfunctional. However, some mutant strains became functional again when they accumulated a total of three extra nucleotides or when they were missing three nucleotides. This rescue effect provided compelling evidence that the genetic code for one amino acid is indeed a three-base, or triplet, code.
