Khorana then used those DNA oligonucleotides as templates for RNA polymerase, and produced specific RNA molecules such as GUGUGUGU. From an mRNA segment such as GUGUGUGU---, there are two alternating codons, GUG and UGU. When employed in in vitro protein synthesis, this mRNA yielded a polypeptide of alternating CYS-VALCYS-VAL.
From the triplet binding assay, Khorana knew that UGU coded for CYS. Therefore, GUG must code for valine (VAL). By constructing these and more complicated definedsequence mRNAs, Khorana was able to verify the entire code.