Parallel Polymer Sequencing Methods

A method of sequencing a target polynucleotide comprising the steps of:

• (a) treating said target nucleotide with an intercalated dye;
• (b) extending a primer annealed to said target polynucleotide utilizing a nucleotide labelled with a label which acts as a FRET partner to said DNA intercalating dye;
• (c) detecting the presence or absence of said nucleotide by means of an imaging technique that utilizes FRET;
• and
• (d) repeating steps (a) through (c);
• wherein steps (a) and (b) can occur in any order.

A method of sequencing a target polynucleotide comprising:

• (a) incorporating a plurality of intercalating dye molecules into the target polynucleotide;
• (b) contacting the target polynucleotide with a solution comprising a polymerase and four types of differently labeled nucleotides, wherein the labels each comprise a fluorescence resonance energy transfer (FRET) partner to the intercalating dye molecules, and wherein the labeled nucleotides each comprise a terminator group;
• (c) incorporating one of the differently labeled nucleotides, using the polymerase, into a chain complementary to the target polynucleotide;
• (d) conducting FRET on the intercalating dye and incorporated differently labeled nucleotide partners, thereby identifying the type of the differently labeled nucleotide incorporated;
• (e) removing the terminator group;
• and (f) repeating steps (a)-(e), thereby sequencing the target polynucleotide.

A method of sequencing a target polynucleotide comprising:

• (a) incorporating a plurality of intercalating dye molecules into the target polynucleotide, wherein the target polynucleotide is disposed on a surface;
• (b) contacting the target polynucleotide with a solution comprising a polymerase and four types of differently labeled nucleotides, wherein the labels each comprise a fluorescence resonance energy transfer (FRET) partner to the intercalating dye molecules, wherein the label each comprise a fluorescent organic dye, and wherein the labeled nucleotides each comprise a terminator group;
• (c) incorporating one of the differently labeled nucleotides by extension from a nick, using the polymerase, into a chain complementary to the target polynucleotide;
• (d) conducting FRET on the intercalating dye and incorporated differently labeled nucleotide partners, thereby identifying the type of the differently labeled nucleotide incorporated;
• (e) providing electromagnetic radiation to the target polynucleotide, thereby removing the terminator group;
• and (f) repeating steps (a)-(e) as a homogeneous or one pot reaction, thereby sequencing the target polynucleotide.