Patent ID: 8609421

Claim:
A method of determining a sequence of amino acids encoded by a target mRNA molecule, comprising: a) providing a reaction mixture comprising the target mRNA molecule, a ribosome, an fMet-tRNA fMet in a P site of the ribosome, and a plurality of types of labeled aminoacyl-tRNAs free in solution, wherein the ribosome and/or the target mRNA molecule is immobilized upon a support such that an observation volume contains no more than one ribosome and/or mRNA molecule, and further wherein the ribosome does not comprise a detectable label or a quenching group, wherein each type of labeled aminoacyl-tRNA comprises a different detectable label that does not undergo FRET with other detectable labels on other reactants in the reaction mixture; b) initiating a processive translation of the mRNA molecule by the ribosome; c) during said processive translation, sequentially and optically detecting association of the ribosome with at least a first labeled aminoacyl-tRNA to produce a first signal, a second labeled aminoacyl-tRNA to produce a second signal, and a third labeled aminoacyl-tRNA to produce a third signal, where said association results in an incorporation of a first amino acid from the first labeled aminoacyl-tRNA, a second amino acid from the second labeled aminoacyl-tRNA, and a third amino acid from the third labeled aminoacyl-tRNA into a nascent polypeptide chain; d) validating the incorporation of the first amino acid into the nascent polypeptide chain, wherein said validating the incorporation of the first amino acid comprises detecting an overlap between the first signal and the second signal; e) validating the incorporation of the second amino acid into the nascent polypeptide chain, wherein said validating the incorporation of the second amino acid comprises detecting an overlap between the second signal and the third signal; and f) identifying the first amino acid and the second amino acid, thereby determining a sequence of amino acids encoded by the target mRNA molecule.