Hybridization probes and methods
First Claim
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1. A method of generating a probe to a nucleic acid target, comprising:
- a) amplifying a plurality of regions of said nucleic acid target that are at least 85% free of undesired sequences wherein said undesired sequences are at least 100 bp in length to generate amplification products in a first amplification, wherein said amplifying comprises a primer pair complementary to said plurality of regions;
b) combining said amplification products to generate a mixture;
c) isolating said amplification products in said mixture;
d) fragmenting said mixture of said isolated amplification products to generate a mixture of fragmented amplification products;
e) size fractionating said fragmented amplification products;
f) ligating said size fractionated amplification products to nucleic acid adaptors wherein said adaptors are each ligated to two or more amplification products, and wherein said adaptors comprise amplification primer sequences;
g) amplifying said amplification products comprising said adaptors to generate amplification products in a second amplification wherein said amplifying comprises a primer pair complementary to said adaptor primer sequences; and
h) labeling said amplification products generated in step g) to generate one or more said probes.
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Abstract
The present invention relates to compositions and methods for detection, analysis, and treatment of nucleic acids. In particular, the present invention relates to compositions and methods for generating and using hybridization probes.
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Citations
14 Claims
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1. A method of generating a probe to a nucleic acid target, comprising:
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a) amplifying a plurality of regions of said nucleic acid target that are at least 85% free of undesired sequences wherein said undesired sequences are at least 100 bp in length to generate amplification products in a first amplification, wherein said amplifying comprises a primer pair complementary to said plurality of regions; b) combining said amplification products to generate a mixture; c) isolating said amplification products in said mixture; d) fragmenting said mixture of said isolated amplification products to generate a mixture of fragmented amplification products; e) size fractionating said fragmented amplification products; f) ligating said size fractionated amplification products to nucleic acid adaptors wherein said adaptors are each ligated to two or more amplification products, and wherein said adaptors comprise amplification primer sequences; g) amplifying said amplification products comprising said adaptors to generate amplification products in a second amplification wherein said amplifying comprises a primer pair complementary to said adaptor primer sequences; and h) labeling said amplification products generated in step g) to generate one or more said probes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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Specification