Methods and compositions for analyzing nucleic acid molecules utilizing sizing techniques
First Claim
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1. A method for determining the identity of a nucleic acid molecule, comprising sequentially:
- (a) generating tagged nucleic acid molecules from one or more selected target nucleic acid molecules, wherein a tag is correlative with a particular nucleic acid fragment and detectable by non-fluorescent spectrometry or potentiometry;
(b) separating said tagged molecules by size;
(c) cleaving said tag from said tagged molecule; and
(d) detecting said tag by non-fluorescent spectrometry or potentiometry, and therefrom determining the identity of said nucleic acid molecule.
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Abstract
Tags and linkers specifically designed for a wide variety of nucleic acid reactions are disclosed, which are suitable for a wide variety of nucleic acid reactions wherein separation of nucleic acid molecules based upon size is required.
271 Citations
40 Claims
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1. A method for determining the identity of a nucleic acid molecule, comprising sequentially:
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(a) generating tagged nucleic acid molecules from one or more selected target nucleic acid molecules, wherein a tag is correlative with a particular nucleic acid fragment and detectable by non-fluorescent spectrometry or potentiometry;
(b) separating said tagged molecules by size;
(c) cleaving said tag from said tagged molecule; and
(d) detecting said tag by non-fluorescent spectrometry or potentiometry, and therefrom determining the identity of said nucleic acid molecule. - View Dependent Claims (3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 33)
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2. A method for detecting a selected nucleic acid molecule, comprising sequentially:
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(a) combining a tagged nucleic acid probe with target nucleic acid molecules under conditions and for a time sufficient to permit hybridization of said tagged nucleic acid probe to a complementary selected target nucleic acid sequence, wherein said tagged nucleic acid probe is detectable by non-fluorescent spectrometry or potentiometry;
(b) increasing or decreasing the size of said hybridized tagged probes, the size of unhybridized probes or target molecules, or the size of the probe;
target hybrids;
(c) separating the tagged probes by size;
(d) cleaving said tag from said tagged probe; and
(e) detecting said tag by non-fluorescent spectrometry or potentiometry, and therefrom detecting said selected nucleic acid molecule. - View Dependent Claims (5, 7)
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17. A method for genotyping a selected organism, comprising sequentially:
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(a) generating tagged nucleic acid molecules from a selected target molecule, wherein a tag is correlative with a particular fragment and may be detected by non-fluorescent spectrometry or potentiometry;
(b) separating said tagged molecules by sequential length;
(c) cleaving said tag from said tagged molecule; and
(d) detecting said tag by non-fluorescent spectrometry or potentiometry, and therefrom determining the genotype of said organism. - View Dependent Claims (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
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18. A method for genotyping a selected organism, comprising sequentially:
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(a) combining a tagged nucleic acid molecule with a selected target molecule under conditions and for a time sufficient to permit hybridization of said tagged molecule to said target molecule, wherein a tag is correlative with a particular fragment and may be detected by non-fluorescent spectrometry or potentiometry;
(b) separating said tagged molecules by sequential length;
(c) cleaving said tag from said tagged molecule; and
(d) detecting said tag by non-fluorescent spectrometry or potentiometry, and therefrom determining the genotype of said organism.
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34. A method for detecting a single selected nucleotide within a nucleic acid molecule, comprising sequentially:
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(a) hybridizing in at least two separate reactions a tagged primer and a target nucleic acid molecule under conditions and for a time sufficient to permit hybridization of said primer to said target nucleic acid molecule, wherein each reaction contains an enzyme which will add a nucleotide chain terminator, and, a nucleotide chain terminator complementary to adenosine, cytosine, guanosine, thymidine or uracil, and wherein each reaction contains a different nucleotide chain terminator;
(b) separating said tagged primers by size;
(c) cleaving said tag from said tagged primer; and
(d) detecting said tag by non-fluorescent spectrometry or potentiometry, and therefrom determining the presence of said selected nucleotide within said nucleic acid molecule. - View Dependent Claims (35, 36, 37, 38, 39, 40)
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Specification