Experimental confirmation that each codon consists of a triplet of nucleotides came in 1961 from studies with acridine dyes---chemical mutagens that cause mutations by inserting themselves into the backbone of DNA double helix. Each inserted dye molecule leads to the insertion of a single nucleotide. Working in Cambridge, England, Francis Crick and Sydney Brenner showed that a single acridine-induced insertion would cause a mutation of the bacteriophage gene that they were studying. A second such insertion would still be nonfunctional, but a third single-base insertion in the same gene restored nearly normal function. The beauty of this experiment was that it was done by observing phenotype alone.
The results clearly supported the hypothesis that each codon consists of three nucleotides. Each insertion, Crick and Brenner reasoned, caused a shift in reading frame, the grouping of nucleotides into codons that specify an amino acid sequence. A shift of one or two nucleotides in a triplet code led to a complete garbling of the message. In contrast, the insertion of three nucleotides restored most of the message's original sense, even though it was slightly different from the original. Crick and Brenner reasoned that a code unit must be a triplet because three nearby insertions seemed to restore the reading frame and confer a nearly normal phenotype.
On the basis of these experiments, molecular biologists concluded that a triplet of nucleotides in DNA specified each amino acid in a protein. Further confirmation came from later studies that showed that three insertions resulted in a polypeptide chain with one extra amino acid, while three deletions resulted in a polypeptide chain missing one amino acid.