Nucleic acid sequencing using affinity reagents

A method for performing a sequencing-by-synthesis reaction, said method comprising the steps of:

• (a) providing a DNA array comprising a plurality of immobilized template nucleic acids comprising a plurality of different template sequences wherein the different template sequences of the are immobilized at different positions on the array;
• (b) annealing oligonucleotide primers to the template nucleic acids wherein the oligonucleotide primers hybridize to predetermined positions on the template nucleic acids;
• (c) combining the template nucleic acids and primers annealed thereto with a polymerase and four different reversible terminator deoxyribonucleotides each reversible terminator deoxyribonucleotide comprising a nucleobase (N), a sugar moiety, and a cleavable blocking group,
• wherein N is adenine (A) or an analog thereof (A′), guanine (G) or an analog thereof (G′), thymine (T) or an analog thereof (T′), and cytosine (C) or an analog thereof (C′),
• wherein at least one of said four different reversible terminator deoxyribonucleotides is unlabeled,
• under conditions in which a plurality of the oligonucleotide primers are extended by incorporation of a single reversible terminator deoxyribonucleotide each to produce a plurality of primer extension products each comprising a 3′ terminus, some of which comprise A or A′ incorporated at the 3′ terminus, some of which comprise T or T′ incorporated at the 3′ terminus, some of which comprise G or G′ incorporated at the 3′ terminus, and some of which comprise C or C′ incorporated at the 3′ terminus;
• (d) contacting the plurality of primer extension products with one or more first affinity reagents under conditions wherein each of said one or more first affinity reagents binds to only one of the four different incorporated reversible terminator deoxyribonucleotides, wherein the first affinity reagents bind to the nucleobase, the sugar moiety, the cleavable blocking group or a combination thereof, of said one of four incorporated reversible terminator deoxyribonucleotides;
• (e) detecting the binding of the one or more first affinity reagents, wherein the binding of a first affinity reagent to a primer extension product comprising an incorporated reversible terminator deoxyribonucleotide identifies the nucleobase of the incorporated reversible terminator deoxyribonucleotide and the nucleobase of the template nucleotide complementary to the nucleobase of the incorporated reversible terminator deoxyribonucleotide.

A method for sequencing a nucleic acid, comprising:

• (a) contacting a nucleic acid template comprising the nucleic acid, a nucleic acid primer complementary to a portion of said template, a polymerase, and an unlabeled RT of Formula I:
• wherein
• R1 is a 3′-O reversible cleavable blocking group;
• R2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), and analogues thereof;
• and
• R3 consists of one or more phosphates;
• under conditions suitable for extending the primer by incorporating the unlabeled RT into a sequence complementary to the nucleic acid template, thereby producing an unlabeled extension product comprising the unlabeled RT;
• (b) contacting the unlabeled extension product with an affinity reagent comprising a detectable label under conditions wherein the affinity reagent binds specifically to the unlabeled RT to produce a labeled extension product comprising the RT;
• and
• (c) identifying the unlabeled RT in the labeled extension product to identify at least a portion of the sequence of said nucleic acid.

A DNA array comprising:

• a plurality of different template DNA molecules immobilized at different locations on the array,
• wherein a plurality of the template DNA molecules comprise primer extension products annealed thereto wherein the primer extension products comprise 3′ reversible terminator deoxyribonucleotides comprising A, T, G or C nucleobases or analogs thereof;
• and
• affinity reagents specifically bound to at least some of the first reversible terminator deoxyribonucleotides.

A kit comprising:

• (a) four types of unlabeled reversible terminator deoxyribonucleotides of Formula I:
• wherein
• R1 is a 3′-O cleavable blocking group;
• R2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), and analogues thereof and each type of reversible terminator deoxyribonucleotide comprises a different nucleobase;
• and
• R3 comprises of one or more phosphates;
• wherein R1 is independently selected for each type of reversible terminator deoxyribonucleotide;
• (b) a plurality of first affinity reagents wherein each first affinity reagent binds a different one of the reversible terminator deoxyribonucleotides when incorporated at the 3′ terminus of a primer extension product;
• and
• (c) packaging for (a) and (b).

A method for detecting a 3′-O-reversible terminator deoxyribonucleotide incorporated at the 3′ end of a primer extension product, wherein the reversible terminator deoxyribonucleotide comprises a nucleobase, a sugar moiety, and a cleavable blocking group, said method comprising

• (a) providing the primer extension product comprising the incorporated reversible terminator deoxyribonucleotide;
• (b) removing the cleavable blocking group of the incorporated reversible terminator deoxyribonucleotide to produce a 3′-OH deoxyribonucleotide moiety of the primer extension product;
• and then
• (c) combining the primer extension product from (b) with a first affinity reagent that binds to the incorporated reversible terminator deoxyribonucleotide, wherein the first affinity reagent binds to the nucleobase and 3′-OH moiety of the 3′-OH primer extension product, and
• (d) detecting binding of the first affinity reagent to the incorporated reversible terminator deoxyribonucleotide.

A method for sequencing a nucleic acid, comprising:

• (a) providing a DNA array comprising (i) a plurality of template DNA molecules, each template DNA molecule comprising a primer binding site and a fragment of the nucleic acid, wherein each of said plurality of template DNA molecules is attached at a position of the array,
• (b) contacting the DNA array with a nucleic acid primer complementary to the primer binding site, a polymerase, and an unlabeled reversible terminator nucleotide (RT) of Formula I:
• wherein
• R1 is a 3′-O cleavable blocking group;
• R2 is a nucleobase selected from adenine (A), cytosine (C), guanine (G), thymine (T), and analogues thereof;
• and
• R3 consists of one or more phosphates;
• under conditions wherein the primer hybridizes to primer binding sites of the template DNA molecules and wherein at least some hybridized primers are extended to incorporate the unlabeled RT into a sequence complementary to the template DNA molecule, thereby producing unlabeled extension products comprising the unlabeled RT;
• (c) contacting the unlabeled extension products with an affinity reagent under conditions wherein the affinity reagent binds specifically to the incorporated RT, wherein the affinity reagent is directly or indirectly associated with a detectable label to produce labeled extension products comprising the RT;
• and
• (d) identifying the RT in the labeled extension products thereby to identify at least a portion of the sequence of said nucleic acid.