Method for replicating an array of nucleic acid probes
First Claim
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1. A method for replicating an array of single-stranded probes on a solid support comprising the steps of:
- a) synthesizing an array of nucleic acids each comprising a non-variant sequence of length C at a 3'"'"'-terminus and a variable sequence of length R at a 5'"'"'-terminus;
b) fixing the array to a first solid support;
c) synthesizing a set of nucleic acids each comprising a sequence complementary to the non-variant sequence;
d) hybridizing the nucleic acids of the set to the array;
e) enzymatically extending the nucleic acids of the set using the variable sequences of the array as templates;
f) denaturing the set of extended nucleic acids; and
g) fixing the denatured nucleic acids of the set to a second solid support to create the replicated array of single-stranded probes.
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Abstract
The invention relates to the replication of probe arrays and methods for replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5'"'"'- and/or 3'"'"'-overhangs.
325 Citations
29 Claims
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1. A method for replicating an array of single-stranded probes on a solid support comprising the steps of:
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a) synthesizing an array of nucleic acids each comprising a non-variant sequence of length C at a 3'"'"'-terminus and a variable sequence of length R at a 5'"'"'-terminus; b) fixing the array to a first solid support; c) synthesizing a set of nucleic acids each comprising a sequence complementary to the non-variant sequence; d) hybridizing the nucleic acids of the set to the array; e) enzymatically extending the nucleic acids of the set using the variable sequences of the array as templates; f) denaturing the set of extended nucleic acids; and g) fixing the denatured nucleic acids of the set to a second solid support to create the replicated array of single-stranded probes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method for replicating an array of single-stranded probes on a solid support comprising the steps of:
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a) fixing a first array of nucleic acids each comprising a non-variant sequence of length C at a 3'"'"'-terminus and a variable sequence of length R at a 5'"'"'-terminus to a first solid support; b) synthesizing a first set of nucleic acids each comprising a sequence complementary to the non-variant sequence; c) hybridizing the nucleic acids of the first set to the first array; d) enzymatically extending the nucleic acids of the first set using the variable sequences of the first array as templates; e) denaturing the first set of extended nucleic acids; f) fixing the denatured nucleic acids of the first set to a second solid support to create the replicated array of single-stranded probes; and g) hybridizing the replicated array with a second set of nucleic acids complementary to the constant sequence of the replicated array to create a partially double-stranded replicated array. - View Dependent Claims (16, 17, 18, 19, 20, 21)
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22. A method for replicating an array of single-stranded probes on a solid support comprising the steps of:
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a) fixing an array of nucleic acids each comprising a non-variant sequence of length C at a 3'"'"'-terminus and a variable sequence of length R at a 5'"'"'-terminus to a first solid support; b) synthesizing a set of nucleic acids each comprising a sequence complementary to the non-variant sequence; c) hybridizing the nucleic acids of the set to the array; d) enzymatically extending the nucleic acids of the set using the variable sequences of the array as templates; e) denaturing the set of extended nucleic acids; and f) fixing the denatured nucleic acids of the set to a second solid support with a streptavidin-biotin or an avidin-biotin bond to create the replicated array of single-stranded probes. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29)
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Specification