Replica amplification of nucleic acid arrays
First Claim
1. A method of making an immobilized nucleic acid molecule array comprising:
- a) providing an immobilized array of spots of a nucleic acid capture activity wherein;
i) said spots are separated by a distance greater than the diameter of said spots; and
ii) the size of said spots is less than the diameter of the excluded volume of said nucleic acid molecule to be captured; and
b) contacting said array of spots of a nucleic acid capture activity with an excess of nucleic acid molecules capable of being bound by said nucleic acid capture activity, said nucleic acid molecules having an excluded volume diameter greater than the diameter of said spots, resulting in an immobilized nucleic acid array in which each said spot of said nucleic acid capture activity can bind only one of said nucleic acid molecules having an excluded volume greater than the size of said spots.
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Accused Products
Abstract
Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.
76 Citations
67 Claims
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1. A method of making an immobilized nucleic acid molecule array comprising:
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a) providing an immobilized array of spots of a nucleic acid capture activity wherein;
i) said spots are separated by a distance greater than the diameter of said spots; and
ii) the size of said spots is less than the diameter of the excluded volume of said nucleic acid molecule to be captured; and
b) contacting said array of spots of a nucleic acid capture activity with an excess of nucleic acid molecules capable of being bound by said nucleic acid capture activity, said nucleic acid molecules having an excluded volume diameter greater than the diameter of said spots, resulting in an immobilized nucleic acid array in which each said spot of said nucleic acid capture activity can bind only one of said nucleic acid molecules having an excluded volume greater than the size of said spots. - View Dependent Claims (2, 3, 4, 5)
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6. A method for the detection of a nucleic acid on an array of nucleic acid molecules, said method comprising:
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a) generating a plurality of a nucleic acid molecule array wherein the nucleic acid molecules of each member of said plurality occupy positions which correspond to those positions occupied by the nucleic acid molecules of each other member of said plurality of a nucleic acid array; and
b) subjecting one or more members of said plurality, but at least one less than the total number of said plurality to a method of signal detection comprising a signal amplification method which renders said member of said plurality of a nucleic acid array non-reusable. - View Dependent Claims (7, 8, 9)
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10. A method of preserving the resolution of nucleic acid features on a first immobilized array during cycles of array replication, said method comprising the following steps:
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a) amplifying the features of a first array to yield an array of features with a hemispheric radius, r, and a cross-sectional area, q, at the surface supporting said array, such that said features remain essentially distinct;
b) contacting said array of features with a radius, r, with a support, maintained at a fixed distance from said first array, said fixed distance less than r, and such that the cross-sectional area of the hemispheric feature, measured at said fixed distance from the surface supporting said first array is less than q, and such that at least a subset of nucleic acid molecules produced by said amplifying are transferred to said support;
c) covalently affixing said nucleic acid molecules to said support to form a replica of said first immobilized array, wherein the positions of said nucleic acid molecules on said replica correspond to the positions of said nucleic acid molecules of said first array from which they were amplified, and wherein the areas occupied on the surface of said support by the individual features of said replica are less than the areas occupied on the surface supporting said first immobilized array. - View Dependent Claims (11, 12)
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13. A method for determining the nucleotide sequence of the features of an immobilized nucleic acid array, said method comprising the steps of:
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a) ligating a first double-stranded nucleic acid probe to one end of a nucleic acid of a feature of said array, said first double stranded nucleic acid probe having a restriction endonuclease recognition site for a restriction endonuclease whose cleavage site is separate from its recognition site and which generates a protruding strand upon cleavage;
b) identifying one or more nucleotides at the end of said polynucleotide by the identity of the first double stranded nucleic acid probe ligated thereto or by extending a strand of the polynucleotide or probe;
c) amplifying the features of said array using a primer complementary to said first double stranded nucleic acid probe, such that only molecules which have been successfully ligated with said first double stranded nucleic acid probe are amplified to yield an amplified array;
d) contacting said amplified array with support such that at least a subset of nucleic acid molecules produced by said amplifying are transferred to said support;
e) covalently attaching said subset of nucleic acid molecules transferred in step (d) to said support to form a replica of said amplified array;
f) cleaving the nucleic acid features of the array with a nuclease recognizing said nuclease recognition site of said probe such that the nucleic acid of the features is shortened by one or more nucleotides; and
g) repeating steps (a)-(f) until the nucleotide sequences of the features of said array are determined. - View Dependent Claims (14, 15, 16, 17, 18, 19)
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20. A method of determining the nucleotide sequence of the features of an array of immobilized nucleic acids comprising the steps of:
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a) adding a mixture comprising an oligonucleotide primer and a template-dependent polymerase to an array of immobilized nucleic acid features under conditions permitting hybridization of the primer to the immobilized nucleic acids;
b) adding a single, fluorescently labeled deoxynucleoside triphosphate to the mixture under conditions which permit incorporation of the labeled deoxynucleotide onto the 3′
end of the primer if it is complementary to the next adjacent base in the sequence to be determined;
c) detecting incorporated label by monitoring fluorescence;
d) repeating steps (b)-(c) with each of the remaining three labeled deoxynucleoside triphosphates in turn; and
e) repeating steps (b)-(d) until the nucleotide sequence is determined. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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35. A method of determining the nucleotide sequence of the features of an array of immobilized nucleic acids comprising the steps of:
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a) adding a mixture comprising an oligonucleotide primer and a template-dependent polymerase to an array of immobilized nucleic acid features under conditions permitting hybridization of the primer to the immobilized nucleic acids;
b) adding a first mixture of three unlabeled deoxynucleoside triphosphates under conditions which permit incorporation of deoxynucleotides to the end of the primer if they are complementary to the next adjacent base in the sequence to be determined;
c) adding a second mixture of three unlabeled deoxynucleoside triphosphates, said second mixture comprising the deoxynucleoside triphosphate not included in the mixture of step (b), under conditions which permit incorporation of deoxynucleotides to the end of the primer if they are complementary to the next adjacent base in the sequence to be determined;
d) repeating steps (b)-(c) for a predetermined number of cycles;
e) adding a single, fluorescently labeled deoxynucleoside triphosphate to the mixture under conditions which permit incorporation of the labeled deoxynucleotide onto the 3′
terminus of the primer if it is complementary to the next adjacent base in the sequence to be determined;
f) detecting incorporated label by monitoring fluorescence;
g) repeating steps (e)-(f), with each of the remaining three labeled deoxynucleoside triphosphates in turn; and
h) repeating steps (e)-(g) until the nucleotide sequence is determined. - View Dependent Claims (36, 37, 38, 39, 40, 41, 42, 43)
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44. A method of determining the nucleotide sequence of the features of a micro-array of nucleic acid molecules, said method comprising the following steps:
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a) creating a micro-array of nucleic acid features in a linear arrangement within and along one side of a polyacrylamide gel, said gel further comprising one or more oligonucleotide primers, and a template-dependent polymerizing activity;
b) amplifying the microarray of step (a);
c) adding a mixture of deoxynucleoside triphosphates, said mixture comprising each of the four deoxynucleoside triphosphates dATP, dGTP, dCTP and dTTP, said mixture further comprising chain-terminating analogs of each of the deoxynucleoside triphosphates dATP, dGTP, dCTP and dTTP, and said chain-terrninating analogs each distinguishably labeled with a spectrally distinguishable fluorescent moiety;
d) incubating said mixture with said micro-array under conditions permitting extension of said one or more oligonucleotide primers;
e) electrophoretically separating the products of said extension within said polyacrylamide gel; and
f) determining the nucleotide sequence of the features of said micro-array by detecting the fluorescence of the extended, terminated and separated reaction products within the gel. - View Dependent Claims (45, 46, 47, 48)
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49. A method of simultaneously amplifying a plurality of nucleic acids, said method comprising the steps of:
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a) creating a micro-array of immobilized oligonucleotide primers;
b) incubating the microarray of step (a) with amplification template and a non-immobilized oligonucleotide primer under conditions allowing hybridization of said template with said oligonucleotide primers;
c) incubating the hybridized primers and template of step (b) with a DNA polymerase activity, and deoxynucleotide triphosphates under conditions permitting extension of the primers;
d) repeating steps (b) and (c) for a defined number of cycles to yield a plurality of amplified DNA molecules. - View Dependent Claims (50, 51, 52, 53, 54, 55)
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56. A method of making a nucleic acid molecule array comprising:
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a) providing a liquid mixture of template nucleic acids, at least one oligonucleotide primer, wherein the at least one oligonucleotide primer includes a linker moiety, and monomers capable of forming a polymerized gel matrix;
b) contacting the mixture of step (a) with a solid support, c) forming a polymerized gel matrix with the linker moiety covalently bound thereto; and
d) amplifying the template nucleic acid to generate a nucleic acid molecule array. - View Dependent Claims (57, 58, 59, 60, 61)
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62. A method of making a plurality of nucleic acid molecule arrays comprising:
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a) providing a first liquid mixture of template nucleic acid, at least one oligonucleotide primer, wherein the at least one oligonucleotide primer includes a linker moiety, and monomers capable of forming a polymerized gel matrix;
b) contacting the mixture of step (a) with a solid support, c) forming a first layer of a polymerized gel matrix with the linker moiety covalently bound thereto, d) providing a second liquid mixture of at least one oligonucleotide primer and monomers capable of forming a polymerized gel matrix, e) contacting the first layer with the second liquid mixture, f) forming a second layer of a polymerized gel matrix, g) amplifying the template nucleic acid and transferring amplified nucleic acid to the second layer, h) removing the second layer; and
i) optionally repeating steps d through h. - View Dependent Claims (63, 64, 65, 66, 67)
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Specification