Apparatus for single molecular sequencing and method of sequencing nucleic acid molecules

A method of sequencing nucleic acid molecules comprising:

• (a) providing at least one nanowell, wherein the at least one nanowell has an observation zone;
• (b) immobilizing a plurality of types of nucleic acid fragments to the observation zone through a plurality of nucleic acid immobilization moieties;
• (c) providing only one primer and a plurality of labelled nucleotide analogs into the at least one nanowell, wherein the only one primer and one of the plurality of types of nucleic acid fragments form a primer-nucleic acid fragment complex in the observation zone;
• (d) forming a nascent strand by initiating a nascent strand synthesizing reaction of the primer-nucleic acid fragment complex through using a polymerase and incorporating the plurality of labelled nucleotide analogs into the primer-nucleic acid fragment complex;
• (e) determining the sequence of one of the plurality of types of the nucleic acid fragments by detecting the incorporated sequence of the plurality of labelled nucleotide analogs;
• (f) dissociating the nascent strand;
• and
• (g) repeating step (c) to step (f).