SYSTEMS AND METHODS FOR HIGH THROUGHPUT, HIGH FIDELITY, SINGLE MOLECULE NUCLEIC ACID SEQUENCING USING TIME MULTIPLEXED EXCITATION

摘要

A method is disclosed of using microscopy in high throughput sequencing of a target nucleic acid. The method includes the steps of disposing in a microreactor a mixture in solution phase comprising a copy of a target nucleic acid, providing a first illumination beam having a center frequency of ?i that is selected to effect fluorescence of a first label associated with a first nucleotide, providing a second illumination beam having a center frequency of ct^ that is selected to effect fluorescence of a second label associated with a second nucleotide, and modulating the first illumination beam and the second illumination beam such that a multiplexed excitation beam is provided that includes illumination from the first illumination beam and the second illumination beam. The method also includes the steps of directing and focusing the multiplexed excitation beam toward a common focal volume, and detecting radiation at an array detector from within the common focal volume. The radiation is associated with the incorporation of an individual nucleotide during template-dependent replication of a copy of the target nucleic acid to achieve high throughput sequencing of the target nucleic acid.