Method and reagent combination for determining nucleotide sequences

Claim 1

A method for determining the sequence of a target nucleic acid polymer comprising: (a) combining a sample containing the target nucleic acid polymer with a mixture of nucleoside triphosphates, a nucleic acid polymerase, and first and second amplification primers, each amplification primer comprising an oligonucleotide having a region that is complementary to the hybridizes with a different strand of the target nucleic acid polymer and being effective as a primer for nucleic acid polymerization by the nucleic acid polymerase to form an amplification mixture, wherein said first amplification primer further comprises a first attachment moiety bonded to the oligonucleotide; (b) repeatedly cycling the amplification mixture between a first condition under which DNA polymerization occurs and a second condition under which denaturation of double stranded DNA to single stranded DNA occurs for a number of cycles sufficient to produce copies of the target nucleic acid polymer in a quantity suitable for use in a DNA sequencing procedure to form an amplified sample comprising copies of the target nucleic acid sequence bonded to the attachment moiety; (c) adding to the amplified sample a plurality of first supports, each comprising a solid matrix and a first attachment site which is capable of immobilizing one strand of the target nucleic acid polymer through the first attachment moiety; (d) allowing the first attachment sites to interact with the first attachment moieties such that the copies of the target nucleic acid in the amplified sample are immobilized on the first supports; (e) separating the immobilized nucleic acids from the amplification mixture; (f) denaturing the amplified nucleic acid sample to form single stranded nucleic acids; and (g) determining the sequence of the single stranded nucleic acids.

Claim 2

A method for determining the sequence of a target nucleic acid polymer comprising: (a) combining a sample containing the target nucleic acid polymer with a mixture of nucleoside triphosphates, a nucleic acid polymerase, and first and second amplification primers, each amplification primer comprising an oligonucleotide having a region that is complementary to the hybridizes with a different strand of the target nucleic acid polymer and being effective as a primer for nucleic acid polymerization by the nucleic acid polymerase to form an amplification mixture, wherein said first amplification primer further comprises a first attachment moiety bonded to the oligonucleotide; (b) repeatedly cycling the amplification mixture between a first condition under which DNA polymerization occurs and a second condition under which denaturation of double stranded DNA to single stranded DNA occurs for a number of cycles sufficient to produce copies of the target nucleic acid polymer in a quantity suitable for use in a DNA sequencing procedure to form an amplified sample comprising copies of the target nucleic acid sequence bonded to the attachment moiety; (c) adding to the amplified sample a plurality of first supports, each comprising a solid matrix and a first attachment site which is capable of immobilizing one strand of the target nucleic acid polymer through the first attachment moiety; (d) allowing the first attachment sites to interact with the first attachment moieties such that the copies of the target nucleic acid in the amplified sample are immobilized on the first supports; (e) separating the immobilized nucleic acids from the amplification mixture; (f) denaturing the amplified nucleic acid sample to form single stranded nucleic acids; and (g) determining the sequence of the single stranded nucleic acids.

Claim 5

A method according to any one of claims 1 to 3, wherein said first attachment moiety and said first attachment site are components of an affinity pair.

Claim 7

A method according to any one of claims 1 to 5, wherein said second amplification primer comprises a second attachment moiety different from said first attachment moiety.

Claim 11

A method according to any one of claims 6 to 9, wherein said first and second supports are concurrently present in the amplified sample and are selected so as to be physically separable from each other.

Claim 14

A method according to any one of claims 1 to 12, wherein the sequence-determination process includes the steps of combining the single-stranded immobilized nucleic acids with a sequencing primer and a sequencing mixture comprising nucleoside triphosphates, one chain terminating nucleotide, a polynucleotide synthesizing enzyme and a labeling moiety which is incorporated during nucleic acid synthesis; allowing polymerization to occur for a period of time sufficient for enough chain termination events to occur to permit determination of the sequence to form a partially polymerized sample; adding a completion reagent comprising a mixture of nucleoside triphosphates to the partially polymerized sample; allowing polymerization to occur in the completion reagent for a period of time sufficient to substantially complete DNA synthesis; separating the completely polymerized sample from the completion reagent; releasing the polymerized nucleic acids from the support; denaturing the polymerized nucleic acids to render them single stranded; analyzing the size of the single stranded nucleic acid polymers to determine the sequence of the target nucleic acid polymer.

Claim 16

A method according to any one of claims 1 to 14, further comprising the step of incubating the amplification mixture at a temperature selected to promote annealing of nucleic acids before each polymerization step.

Claim 32

The method according to anyone of claims 1 to 15 for de­termining in the target nucleic acid polymer at least one potential nucleotide variation.