Method and apparatus for performing amplification of nucleic acid on supports
First Claim
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1. A method of amplifying a single stranded first nucleic acid in a sample potentially containing said first nucleic acid, said method comprising:
- (a) forming an immersion product, said immersion product comprising said sample and at least one primer set of nucleic acids, said set of nucleic acids comprising at least one second nucleic acid and at least one third nucleic acid, said second nucleic acid and said third nucleic acid being covalently affixed to a support and separated on said support by a distance less than the length of said first nucleic acid, said second nucleic acid being capable of forming a hybridization product with said first nucleic acid, said third nucleic acid being capable of forming a hybridization product with a nucleic acid that is complementary to said first nucleic acid;
(b) forming a hybridization product comprising said first nucleic acid and said second nucleic acid, in the event said first nucleic acid is present, by imposing hybridization conditions on the immersion product;
(c) forming a first amplification product comprising said second nucleic acid extended by nucleotides complementary to said first nucleic acid, said first amplification product being capable of forming a hybridization product with said third nucleic acid;
(d) denaturing the first amplification product from said first nucleic acid;
(e) forming a second hybridization product comprising said first amplification product and said third nucleic acid, by imposing hybridization conditions on the immersion product; and
(f) forming a second amplification product comprising said third nucleic acid extended by nucleotides complementary to said first amplification product, wherein said first amplification product provides the template for said second amplification product, and said third nucleic acid and said template are immobilized on a common support.
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Abstract
This invention features methods and apparatus for performing nucleic acid hybridization and amplification processes on a support. Such methods and apparatus are useful for synthesizing nucleic acid and detecting target nucleic acid for diagnostics and therapeutics.
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Citations
18 Claims
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1. A method of amplifying a single stranded first nucleic acid in a sample potentially containing said first nucleic acid, said method comprising:
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(a) forming an immersion product, said immersion product comprising said sample and at least one primer set of nucleic acids, said set of nucleic acids comprising at least one second nucleic acid and at least one third nucleic acid, said second nucleic acid and said third nucleic acid being covalently affixed to a support and separated on said support by a distance less than the length of said first nucleic acid, said second nucleic acid being capable of forming a hybridization product with said first nucleic acid, said third nucleic acid being capable of forming a hybridization product with a nucleic acid that is complementary to said first nucleic acid;
(b) forming a hybridization product comprising said first nucleic acid and said second nucleic acid, in the event said first nucleic acid is present, by imposing hybridization conditions on the immersion product;
(c) forming a first amplification product comprising said second nucleic acid extended by nucleotides complementary to said first nucleic acid, said first amplification product being capable of forming a hybridization product with said third nucleic acid;
(d) denaturing the first amplification product from said first nucleic acid;
(e) forming a second hybridization product comprising said first amplification product and said third nucleic acid, by imposing hybridization conditions on the immersion product; and
(f) forming a second amplification product comprising said third nucleic acid extended by nucleotides complementary to said first amplification product, wherein said first amplification product provides the template for said second amplification product, and said third nucleic acid and said template are immobilized on a common support. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18)
(a) amplifying said first nucleic acid according to the amplification method of claim 16; and
(b) monitoring said support for the presence of one of said amplification products, which presence is indicative of the presence of said first nucleic acid and which absence is indicative of the absence of said first nucleic acid.
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16. The method of claim 1, wherein said second nucleic acid and said third nucleic acid comprise different nucleotide sequences.
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18. A method for detecting the presence or absence of a first nucleic acid in a sample potentially containing said first nucleic acid, said method comprising:
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(a) amplifying said first nucleic acid according to the amplification method of claim 1; and
(b) monitoring said support for the presence of one of said amplification products, which presence is indicative of the presence of said first nucleic acid and which absence is indicative of the absence of said first nucleic acid.
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17. A method of mapping a target nucleic acid in a sample, said method comprising:
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(a) providing multiple copies of a first nucleic acid and dividing said target nucleic acid into a plurality of segments having overlapping sequences;
(b) providing a plurality of primer sets of nucleic acid, each primer set comprising at least a second nucleic acid and a third nucleic acid, said second nucleic acid and said third nucleic acid being covalently affixed to a support and separated on said support by a distance less than a first segment of said first nucleic acid, said second nucleic acid being capable of forming a hybridization product with a segment of said first nucleic acid, said third nucleic acid being complementary to the antisense strand of said segment, each primer set having second and third nucleic acids directed to a segment and separate from other primer sets of nucleic acids;
(c) forming an immersion product comprising the support having a plurality of primer sets of nucleic acid and a first nucleic acid segment;
(d) forming a hybridization product comprising said segment and said second nucleic acid of each set, in the event said segment is present, by imposing hybridization conditions on the immersion product;
(e) forming a first amplification product comprising said second nucleic acid extended by nucleotide complementary to said segment of said first nucleic acid, said first amplification product being capable of forming a hybridization product with said third nucleic acid;
(f) denaturing the first amplification product from said first nucleic acid segment;
(g) forming a second hybridization product comprising said first amplification product and said third nucleic acid, by imposing hybridization conditions on the immersion product;
(h) forming a second amplification product comprising said third nucleic acid extended by nucleotides complementary to said first amplification product; and
(i) monitoring said support for the presence of one of said amplification products, which presence is indicative of the presence of the segment and which absence is indicative of the absence of said segment, said segments which form amplification products at identical sets indicating the presence of overlapping sequences of the segments.
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Specification