SEQUENCE-SPECIFIC LARGE VOLUME SAMPLE PREPARATION METHOD AND ASSAY
First Claim
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1. A large volume sample preparation method, the method comprising:
- (a) suspending a biological sample in about 1 mL or more of a collection media;
(b) denaturing and lysing the biological sample by adding a denaturation agent and lysis buffer to the suspended biological sample;
(c) hybridizing a target nucleic acid molecule to at least one polynucleotide probe;
(d) capturing the hybridized target nucleic acid molecule on a support;
wherein the denaturing and lysing step (b) is complete in less than about 10 minutes and the combination of the hybridizing step (c) and the capturing step (d) is complete in less than about 25 minutes and 10 copies or more of the target nucleic acid molecule are isolated in less than about 1 hour.
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Abstract
Methods of selectively and rapidly identifying target nucleic acid molecules in large volumes of collection media where the target is present in a low concentration are disclosed. The methods can be used to identify, isolate, purify, or enrich a nucleic acid molecule containing a specific target sequence from a sample of nucleic acid molecules that do not contain the specific target sequence. Once isolated, the nucleic acid molecule containing a specific target sequence may be amplified or used in a variety of detection assays.
106 Citations
24 Claims
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1. A large volume sample preparation method, the method comprising:
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(a) suspending a biological sample in about 1 mL or more of a collection media; (b) denaturing and lysing the biological sample by adding a denaturation agent and lysis buffer to the suspended biological sample; (c) hybridizing a target nucleic acid molecule to at least one polynucleotide probe; (d) capturing the hybridized target nucleic acid molecule on a support; wherein the denaturing and lysing step (b) is complete in less than about 10 minutes and the combination of the hybridizing step (c) and the capturing step (d) is complete in less than about 25 minutes and 10 copies or more of the target nucleic acid molecule are isolated in less than about 1 hour. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for detecting the presence of a target nucleic acid molecule in a large sample volume, the method comprising:
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(a) suspending the biological sample in about 1.0 mL or more of a collection media or obtaining a biological sample in urine, blood, or serum; (b) denaturing the target nucleic acid molecule in the biological sample; (c) forming a double-stranded nucleic acid hybrid by contacting at least one polynucleotide probe with the target nucleic acid molecule; (d) forming a double-stranded nucleic acid hybrid-support complex by capturing the double-stranded nucleic acid hybrid on a support; wherein 10 copies or more of the target nucleic acid molecule are capable of being identified in about 30 minutes to about 3 hours. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23)
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24. A sample preparation method, the method comprising:
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(a) suspending a biological sample in about 100 μ
l or more of a collection media;(b) denaturing and lysing the biological sample by adding a denaturation agent and lysis buffer to the suspended biological sample; (c) hybridizing a target nucleic acid molecule to at least one polynucleotide probe; (d) capturing the hybridized target nucleic acid molecule on a support; wherein the denaturing and lysing step (b) is complete in less than about 10 minutes and the combination of the hybridizing step (c) and the capturing step (d) is complete in less than about 25 minutes and 10 copies or more of the target nucleic acid molecule are isolated in less than about 1 hour.
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Specification