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Method for producing copies of a nucleic acid using immobilized oligonucleotides

  • US 5,922,574 A
  • Filed: 11/22/1995
  • Issued: 07/13/1999
  • Est. Priority Date: 05/28/1994
  • Status: Expired due to Term
First Claim
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1. A method of producing copies of at least part of a target nucleic acid strand present or potentially present in a sample, the method comprising the steps of(i) providing a solid support system having covalently linked thereon a plurality of single stranded oligonucleotides which are capable of hybridizing to the target strands,(ii) providing the target strands in single stranded form and hybridizing the target strands to oligonucleotides on the support, the number of oligonucleotides exceeding the number of target strands by at least a factor of 10,(ii)(a) washing the support system to remove non-hybridized material,(iii) producing copy target 1 strands which incorporate the covalently linked oligonucleotides and include a nucleic acid sequence complementary to at least part of the target strand, wherein the production of said copy target 1 strands comprises a primer extension step in which the target strand is used as a template to generate at least part of the copy target 1 sequence,(iv) denaturing the target strands hybridized to the copy target 1 strands,(v) simultaneously or sequentially in either order,(a) hybridizing primers to the copy target 1 strands immobilized on the solid support, and(b) hybridizing the denatured target strands to oligonucleotides on the support which have not been converted to copy target 1 strands,(vi) simultaneously,(a) producing copy target 2 strands which comprise a nucleic acid sequence of interest from the primers hybridized to the copy target 1 strands, wherein the production of copy target 2 strands comprises a primer extension step;

  • and(b) producing further copy target 1 strands from the target strands hybridized to oligonucleotides on the support, wherein the production of further copy target 1 strands comprises a primer extension step;

    (vii) denaturing the copy target 2 strands hybridized to the copy target 1 strands and rehybridizing the copy target 2 strands to oligonucleotides on the support which have not been converted to copy target 1 strands,(viii) hybridizing primers to the copy target 1 strands immobilized on the solid support,(ix) simultaneously,(a) producing copy target 2 strands which comprise the nucleic acid sequence of interest from the primers hybridised to the copy target 1 strands, wherein the production of copy target 2 strands comprises a primer extension step; and

    (b) producing further copy target 1 strands from copy target 2 strands hybridized to the oligonucleotides on the support, wherein the production of further copy target 1 strands comprises a primer extension step; and

    (x) repeating steps (vii)-(ix) as many times as required in order to produce copies of at least part of the target nucleic acid strand.

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