Method for producing copies of a nucleic acid using immobilized oligonucleotides
First Claim
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.
7 Assignments
0 Petitions
Accused Products
Abstract
Amplification of nucleic acids using oligonucleotides immobilized on solid phase supports is disclosed. Target nucleic acid strands (in a sample to be analyzed) are hybridized to the oligonucleotides and copy target strands (incorporating immobilized oligonucleotide) are produced using the target strands as templates. The target strands are denatured from the copy target strands and rehybridized to non-extended oligonucleotides. In the next step, new copy target 1 strands are synthesized from the oligonucleotides hybridized to the target strands, and simultaneously copy target 2 strands are synthesized using previously generated copy target 1 strands. Sequence of denaturing, rehybridizing, and producing copy target 1 and copy target 2 strands is repeated as often as necessary to give the desired degree of amplification.
-
Citations
19 Claims
-
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. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
- and
-
2. 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) rehybridizing the denatured target strands to oligonucleotides on the support which have not been converted to copy target 1 strands and producing therefrom further copy target 1 strands, (vi) hybridizing primers to the copy target 1 strands immobilized on the solid support and producing therefrom copy target 2 strands which comprise a nucleic acid sequence of interest, (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 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 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. - View Dependent Claims (17)
- and
-
3. 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) washing the support system to remove said target strands, (vi) hybridizing primers to the copy target 1 strands immobilized on the solid support and producing therefrom copy target 2 strands which comprise a nucleic acid sequence of interest, and (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 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 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.
- and
-
4. A method of producing copies of at least part of a 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 each of two different single stranded oligonucleotides 0 and 0'"'"' each of which is capable of hybridizing to a respective one of two complementary strands of a double stranded nucleic acid, said complementary stands providing target 1 and 1'"'"' strands, (ii) providing the target 1 and 1'"'"' 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 and 1'"'"' strands each of which incorporates an covalently linked oligonucleotide 0 or 0'"'"' respectively, and a nucleic acid sequence complementary to at least part of the target 1 or 1'"'"' strand respectively, wherein the production of said copy target 1 and 1'"'"' strands comprises a primer extension step in which the respective target strands are used as templates to generate at least part of the copy target 1 or 1'"'"' sequence, (iv) denaturing the target 1 and 1'"'"' strands hybridised to the copy target 1 and 1'"'"' strands and rehybridizing at least some of the target 1 and 1'"'"' strands to oligonucleotides on the support which have not been converted to copy target 1 or 1'"'"' strands, (v) hybridizing primers to the copy target 1 and 1'"'"' strands immobilized on the solid support, (vi) simultaneously, (a) producing copy target 2 and 2'"'"' strands respectively from the primers hybridized to the copy target 1 and 1'"'"' strands, wherein the production of copy target 2 and 2'"'"' strands comprises a primer extension step; - and
(b) producing further copy target 1 and 1'"'"' strands from target 1 and 1'"'"' strands hybridized to the immobilized oligonucleotides, wherein the production of copy target 1 and 1'"'"' strands comprises a primer extension step; (vii) denaturing the copy target 2 and 2'"'"' strands hybridised to the copy target 1 and 1'"'"' strands and rehybridizing the copy target 2 and 2'"'"' strands to oligonucleotides on the support which have not been converted to copy target strands, (viii) hybridizing primers to the copy target 1 and 1'"'"' strands immobilized on the solid support, (ix) simultaneously (a) producing copy target 2 and 2'"'"' strands which comprise the nucleic acid sequence of interest from the primers hybridized to the copy target 1 and 1'"'"' strands, wherein the production of copy target 2 and 2'"'"' strands comprises a primer extension step; and (b) producing further copy target 1 and 1'"'"' strands from the copy target 2 and 2'"'"' strands hybridized to oligonucleotides on the support, wherein the production of copy target 1 and 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.
- and
-
16. 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 strands hybridized to the copy target 1 strands and rehybridising at least some of the target strands to oligonucleotides on the support which have not been converted to copy target 1 strands, (v) hybridizing primers to copy target 1 strands immobilized on the solid support, (vi) simultaneously (a) producing copy target 2 strands which comprise the nucleic acid sequence of interest from the primers hybridized to 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 target strands hybridized to the immobilized oligonucleotides, 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 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 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 copy target 2 strands hybridized to oligonucleotides on the support, wherein the production of further copy target 1 strands comprises a primer extension step; (x) repeating steps (vii)-(ix) as many times as required, (xi) hybridizing primers to the copy target 1 strands at locations such that extension products of the primers using the copy target 1 strands as templates incorporate the sequence of interest, (xii) extending the primers using the copy target 1 strands as templates, and (xiii) denaturing the product strands from the copy target 1 strands and collecting the product strands.
- and
-
18. A method of producing copies of at least part of a 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 each of two different single stranded oligonucleotides 0 and 0'"'"' each of which is capable of hybridizing to a respective one of two complementary strands of a double stranded nucleic acid, said complementary strands providing target 1 and 1'"'"' strands, (ii) providing the target 1 and 1'"'"' 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 and 1'"'"' strands each of which incorporates covalently linked oligonucleotide 0 or 0'"'"' respectively, and a nucleic acid sequence complementary to at least part of the target 1 or 1'"'"' strand respectively, wherein the production of said copy target 1 and 1'"'"' strands comprises a primer extension step in which the respective target strands are used as templates to generate at least part of the copy target 1 or 1'"'"' sequence, (iv) denaturing the target 1 and 1'"'"' strands hybridized to the copy target 1 and 1'"'"' strands and washing the support system to remove the target 1 and 1'"'"' strands, (v) hybridizing primers to the copy target 1 and 1'"'"' strands immobilized on the solid support, (vi) producing copy target 2 and 2'"'"' strands, respectively, from the primers hybridized to the copy target 1 and 1'"'"' strands, wherein the production of copy target 2 and 2'"'"' strands comprises a primer extension step; -
(vii) denaturing the copy target 2 and 2'"'"' strands hybridized to the copy target 1 and 1'"'"' strands and rehybridizing the copy target 2 and 2'"'"' strands to oligonucleotides on the support which have not been converted to copy target strands, (viii) hybridizing primers to the copy target 1 and 1'"'"' strands immobilized on the solid support, (ix) simultaneously, (a) producing copy target 2 and 2'"'"' strands which comprise the nucleic acid sequence of interest from the primers hybridized to the copy target 1 and 1'"'"' strands, wherein the production of copy target 2 and 2'"'"' strands comprises a primer extension step; and (b) producing further copy target 1 and 1'"'"' strands from the copy target 2 and 2'"'"' strands hybridized to oligonucleotides on the support, wherein the production of copy target 1 and 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.
-
-
19. 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 strands hybridized to the copy target 1 strands and washing the support system to remove said target strands, (v) hybridizing primers to copy target 1 strands immobilized on the solid support, (vi) producing copy target 2 strands which comprise the nucleic acid sequence of interest from the primers hybridized to copy target 1 strands, wherein the production of copy target 2 strands comprises a primer extension step, and (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 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 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 copy target 2 strands hybridized to oligonucleotides on the support, wherein the production of further copy target 1 strands comprises a primer extension step; -
(x) repeating steps (vii)-(ix) as many times as required, (xi) hybridizing primers to the copy target 1 strands at locations such that extension products of the primers using the copy target 1 strands as templates incorporate the sequence of interest, (xii) extending the primers using the copy target 1 strands as templates, and (xiii) denaturing the product strands from the copy target 1 strands and collecting the product strands.
-
Specification