Sequencing by emergence

A method of sequencing a nucleic acid, comprising:

• (a) fixing the nucleic acid in double stranded linearized stretched form on a test substrate thereby forming a fixed stretched double stranded nucleic acid;
• (b) denaturing the fixed stretched double stranded nucleic acid to single stranded form on the test substrate thereby obtaining a fixed first strand and a fixed second strand of the nucleic acid, wherein respective bases of the fixed second strand lie adjacent to corresponding complementary bases of the fixed first strand;
• (c) exposing the fixed first strand and the fixed second strand to a respective pool of a respective oligonucleotide probe in a set of oligonucleotide probes, wherein each oligonucleotide probe in the set of oligonucleotide probes is of a predetermined sequence and length, the exposing (c) occurring under conditions that allow for individual probes of the respective pool of the respective oligonucleotide probe to bind and form a respective heteroduplex with portions of the fixed first strand or the fixed second strand that is complementary to the respective oligonucleotide probe thereby giving rise to a respective instance of optical activity;
• (d) measuring a location on the test substrate and a duration of each respective instance of optical activity occurring during the exposing (c) using a two-dimensional imager;
• (e) repeating the exposing (c) and measuring (d) for respective oligonucleotide probes in the set of oligonucleotide probes, thereby obtaining a plurality of sets of positions on the test substrate, each respective set of positions on the test substrate corresponding to an oligonucleotide probe in the set of oligonucleotide probes;
• and
• (f) determining the sequence of at least a portion of the nucleic acid from the plurality of sets of positions on the test substrate by compiling the positions on the test substrate represented by the plurality of sets of positions.