Method for performing amplification of nucleic acid on supports

• US 6,090,592 A
• Filed: 05/29/1997
• Issued: 07/18/2000
• Est. Priority Date: 08/03/1994
• Status: Expired due to Term

• Current Assignee: Illumina Incorporated, MOSAID Technologies Inc. (f/k/a Conversant Intellectual Property Management), MT Technology, Inc., Whitehead Institute for Biomedical Research
• Original Assignee: MOSAID Technologies Inc. (f/k/a Conversant Intellectual Property Management), Whitehead Institute for Biomedical Research
• Inventors: Kron, Stephen J., Adams, Christopher P.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/776,859
• Time in Patent Office: 1,146 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.3, 536/24.31, 536/24.32, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

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.

Claims

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 comprising different nucleic acid sequences and 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.

2. The method of claim 1 wherein said support is an epoxy silane derivatized support.

3. The method of claim 1 wherein said second nucleic acid has a 5'"'"' amino group which 5'"'"' amino group is covalently bonded to hexaethylene glycol functional group covalently bonded to said support.

4. The method of claim 1 wherein said first nucleic acid has a size of about one to about ten kilobases.

5. The method of claim 1 wherein the amplification product is formed by imposing on said immersion product amplification conditions which comprise applying a thermostable polymerase to said hybridization product.

6. The method of claim 1 wherein the amplification product has a label for detection.

7. The method of claim 6 wherein the nucleotides are radiolabeled.

8. The method of claim 6 wherein the nucleotides are labeled with one or more label consisting of the group selected from chemiluminescence, radioactive, luminescent, and fluorescent agents.

9. The method of claim 1 wherein said first and second amplification products are made simultaneously.

10. The method of claim 1 wherein said primer set contains a plurality of second nucleic acids and a plurality of third nucleic acids, said first amplification product is capable of forming a hybridization product with a plurality of third nucleic acids and said second amplification product is capable of forming a hybridization product with a plurality of second nucleic acids, permitting a plurality of first and second amplification products to form upon the presence of first nucleic acids.

11. The method of claim 1 wherein said primer set is confined to a finite area of said support.

13. The method of claim 1, wherein said first nucleic acid comprises one strand of a double-stranded nucleic acid.

14. The method of claim 1, which further includes the steps of (i) denaturing said second amplification product to yield a set of immobilized extension products;

(ii) forming further hybridization products between at least one member of said set of immobilized extension products and at least one primer from another set of said at least one primer sets of nucleic acids affixed to said support; and

(iii) forming a further amplification product comprising said at least one primer extended by nucleotides complementary to said at least one member of said set of immobilized extension products, said steps (i), (ii) and (iii) being carried out a sufficient number of times to achieve the desired level of amplification.

15. The method of claim 14, wherein said steps (i), (ii) and (iii) are carried out a number of times appropriate to exhaust the primer sets of nucleic acid present on said support.

16. The method of claim 1, wherein said second nucleic acid and said third nucleic acid comprise different nucleic acid sequences.

17. 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:

(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.

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:

(a) amplifying said first nucleic acid according to the amplification method of claim 14; 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.

12. A method of mapping a target nucleic acid in a sample, said method comprising:

(a) providing multiple copies of a first nucleic acid and dividing said first 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 comprising different nucleuic acid sequences and 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 product, 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.