Polycationic supports and nucleic acid purification separation and hybridization
First Claim
1. A method for purifying a polynucleotide greater than 100 nucleotides in length from an impure biological sample comprising an oligonucleotide 10 to 100 nucleotides in length comprising the steps of:
- (a) contacting said biological sample with a solid support comprising a plurality of cations selected from the group consisting of ammonium, immonium and guanidinium ions, wherein said polynucleotide binds to said solid support and said oligonucleotide does not bind to said solid support, wherein said polynucleotide is at least three times greater in length than skid oligonucleotide; and
(b) separating said oligonucleotide from said solid support while said polynucleotide is held to said solid support.
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Accused Products
Abstract
Described herein is the use of polycationic solid supports in the purification of nucleic acids from solutions containing contaminants. The nucleic acids non-covalently bind to the support without significant binding of contaminants permitting their separation from the contaminants. The bound nucleic acids can be recovered from the support. Also described is the use of the supports as a means to separate polynucleotides and hybrids thereof with a nucleotide probe from unhybridized probe. Assays for target nucleotide sequences are described which employ this separation procedure.
194 Citations
73 Claims
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1. A method for purifying a polynucleotide greater than 100 nucleotides in length from an impure biological sample comprising an oligonucleotide 10 to 100 nucleotides in length comprising the steps of:
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(a) contacting said biological sample with a solid support comprising a plurality of cations selected from the group consisting of ammonium, immonium and guanidinium ions, wherein said polynucleotide binds to said solid support and said oligonucleotide does not bind to said solid support, wherein said polynucleotide is at least three times greater in length than skid oligonucleotide; and (b) separating said oligonucleotide from said solid support while said polynucleotide is held to said solid support. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 73)
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20. An assay for detecting a target sequence present on a polynucleotide in an impure biological sample comprising the steps of:
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(a) contacting said biological sample with a labelled oligonucleotide probe able to hybridize to said target sequence to form a hybrid; (b) contacting said hybrid with a solid support, said solid support comprising a plurality of cations selected from the group consisting of ammonium, immonium and guanidinium ions, wherein said hybrid binds to said solid support and forms a bound phase and unhybridized oligonucleotide probe is not bound to said solid support and forms a free phase, wherein said hybrid is at least three times greater in size than said oligonucleotide probe; and (c) detecting the presence of said target sequence by quantitatively or qualitatively measuring the label in said bound phase or said free phase. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33)
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34. An assay for detecting a target sequence present on a polynucleotide in an impure biological sample comprising the steps of:
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(a) contacting said biological sample with an oligonucleotide probe able to hybridize to said target sequence to form a hybrid; (b) contacting said hybrid with a solid support, said solid support comprising a plurality of cations selected from the group consisting of ammonium, immonium and guanidinium ions, wherein said hybrid binds to said solid support and unhybridized oligonucleotide probe is not bound to said solid support, wherein said hybrid is at least three times greater in size than said oligonucleotide probe; (c) separating said solid support from said unhybridized oligonucleotide probe; and (d) detecting the presence of said target sequence by quantitatively or qualitatively measuring said oligonucleotide probe which is bound as said hybrid to said solid support or said probe which is not hybridized. - View Dependent Claims (35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
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47. An assay for detecting a target sequence present on a polynucleotide in an impure biological sample comprising the steps of:
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(a) contacting said biological sample with a solid support, said solid support comprising a plurality of cations selected from the group consisting of ammonium, immonium and guanidinium ions, wherein said polynucleotide binds to said solid support to form a bound polynucleotide; (b) providing to said bound polynucleotide a labelled oligonucleotide probe able to hybridize to said target sequence to form a hybrid bound by said solid support, wherein said hybrid is at least three times greater in size than said oligonucleotide probe such that said oligonucleotide probe which is not hybridized is not bound to said solid support; (c) separating said solid support from unhybridized oligonucleotide probe; and (d) detecting the presence of said target sequence by quantitatively or qualitatively measuring said oligonucleotide probe which is bound as said hybrid to said solid support or said probe which is not hybridized. - View Dependent Claims (48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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60. A kit for detecting a target sequence which may be present on a polynucleotide in an impure biological sample comprising:
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(a) an oligonucleotide probe able to form a hybrid with said target sequence; and (b) a solid support comprising a plurality of cations selected from the group consisting of ammonium, immonium and guanidinium ions, wherein said hybrid is at least three times greater in size than said oligonucleotide probe such that said hybrid binds to said solid support and said oligonucleotide probe does not bind to said solid support in said impure biological sample. - View Dependent Claims (61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72)
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Specification