Method and apparatus for performing large numbers of reactions using array assembly with releasable primers

A method for performing a plurality of polynucleotide amplification reactions, comprising the steps of:

• (a) obtaining first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;
• (b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;
• (c) assembling said first and second solid supports, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;
• and
• (d) releasing said primers;
• (e) performing said plurality of polynucleotide amplification reactions.

A method for performing a plurality of polynucleotide amplification reactions and capturing amplification products, comprising the steps of:

• (a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;
• (b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;
• (c) assembling said first and second solid supports, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;
• (d) releasing said primers;
• (e) generating amplification products of said polynucleotide amplification reactions;
• and
• (f) capturing said amplified products by a plurality of immobilized polynucleotide probes on either said first or second solid support through hybridization.

A method for detecting a plurality of polynucleotide sequence variations, comprising the steps of:

• (a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;
• (b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;
• (c) assembling said first and second solid support, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;
• (d) releasing said primers;
• (e) generating amplification products of said polynucleotide amplification reactions;
• (f) capturing said amplified products by a plurality of immobilized polynucleotide probes on either said first or second array through hybridization;
• and
• (g) detecting polynucleotide sequence variations by hybridization complexes in step (f).

A method for quantitating polynucleotides in a target nucleic acid, comprising the steps of:

• (a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of finite areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;
• (b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each area of said second solid support;
• (c) assembling said first and second solid support, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;
• (d) releasing said primers;
• (e) generating amplification products of said polynucleotide amplification reactions;
• and
• (f) quantitating amplified products.

A method for detecting polynucleotide sequence variations in a target nucleic acid, comprising the steps of:

• (a) obtaining a first solid support wherein a plurality of immobilized moieties are confined to a plurality of areas on the surface of said first solid support and each said immobilized moiety contains a releasable site and a primer;
• (b) obtaining a second solid support wherein the surface of said second solid support contains a plurality of areas and reactants of said polynucleotide amplification reactions are confined on each areas of said second solid support;
• (c) assembling said first and second solid supports, wherein said reactants of said polynucleotide amplification reactions on said second solid support are in contact with said immobilized moieties on said first solid support;
• (d) releasing said primers;
• (e) generating amplification products of said polynucleotide amplification reactions;
• and
• (f) capturing said amplified products by a plurality of immobilized polynucleotide probes on either first or second array though hybridization;
• and
• (g) detecting polynucleotide sequence variations by a polynucleotide modifying enzyme.

A method for amplifying a target nucleic acid, capturing the amplified product, and detecting a polynucleotide sequence variation in the amplified product, comprising the steps of:

• (a) obtaining a first solid support wherein:
• (1) the surface of said first solid support comprises a first, second, third, and fourth areas;
• (2) a first chemical moiety, comprising a releasable forward primer for said target nucleic acid, is immobilized on said first area;
• (3) a second chemical moiety, comprising a releasable reverse primer for said target nucleic acid, is immobilized on said second area;
• (4) a first polynucleotide probe, comprising a subsequence complementary to one variant of said polynucleotide variation, is immobilized on said third area said subsequence containing at least one interrogation position complementary to a corresponding nucleotide in said variant;
• and
• (5) a second polynucleotide probe is immobilized to said fourth area, said second polynucleotide probe differing from said first polynucleotide probe by at least one nucleotide;
• (b) obtaining a second solid support wherein the surface of said solid support comprises a reaction well and a mixture of reacts comprising a DNA polymerase, said target nucleic acid, and deoxynucleotides are placed within said reaction well;
• (c) assembling said first and second solid support, wherein said mixture of reactants are in contact with said first, second, third, and fourth areas on said first solid support;
• (d) releasing said releasable forward and reverse primers;
• (e) generating the amplified product for said target nucleic acid;
• (f) capturing the amplified product by said first or second polynucleotide probe through hybridization;
• (g) washing said first solid support;
• (h) comparing the relative binding of two probes on said first solid support;
• and
• (i) identifying said polynucleotide variation in the amplified product.

A method for amplifying a target nucleic acid, capturing the amplified product, and detecting a polynucleotide sequence variation in the amplified product, comprising the steps of:

• (a) obtaining a first solid support wherein:
• (1) the surface of said first array comprises a first, second, third, and fourth areas;
• (2) a first chemical moiety, comprising a releasable forward primer specific for said region of said target nucleic acid, is immobilized on said first area;
• (3) a second chemical moiety, comprising a releasable reverse primer specific for said region of said target nucleic acid, is immobilized on said second area;
• (4) a first polynucleotide probe, comprising a subsequence complementary to one variant of said polynucleotide variation, is immobilized on said third area, said subsequence containing at least one interrogation position complementary to a corresponding nucleotide in said variant;
• and
• (5) a second polynucleotide probe is immobilized to said fourth area, said second probe differing from said first probe by at least one nucleotide;
• (b) obtaining a second solid support wherein the surface of said solid support comprises a reaction well and a mixture of reactants comprising a DNA polymerase, said target nucleic acid, and deoxynucleotides are placed within said reaction well;
• (c) assembling said first and second solid support, wherein said mixture of reactants are in contact with said first, second, third, and fourth areas on said first solid support;
• (d) releasing said releasable forward and reverse primers;
• (e) generating the amplified product for said target nucleic acid;
• (f) capturing the amplified product by said first or second polynucleotide probes through hybridization;
• (g) extending said one or more hybridization complexes in step (f);
• (h) washing said first solid support;
• and
• (i) identifying said polynucleotide variation using said one or more extended products in step (g).