Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom
First Claim
1. A method for comparing relative amounts of specific nucleic acid molecules in at least two samples, comprising the steps of:
- (a) generating a target pool comprising a first and a second sample, wherein said first sample comprises nucleic acid molecules of a first source, and said first source nucleic acid molecules are linked to a first label, and wherein said second sample comprises nucleic acid molecules of a second source, and said second source nucleic acid molecules are linked to a second label;
(b) contacting said target pool with a plurality of solid supports each having attached thereto multiple capture oligonucleotides of a unique sequence under conditions which promote the formation of perfectly matched duplexes between said capture oligonucleotides and nucleic acid molecule complements within said target pool; and
(c) sorting the solid supports according to the relative amount of said first label and said second label;
wherein the unique capture oligonucleotides attached to each solid support comprise a stretch of from about 10 to about 40 nucleotides of random sequence, or a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from 7 to about 15 nucleotides.
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Abstract
The present invention relates to a method for the comparative assessment of the level of specific nucleic acid sequences in samples derived from different sources. More specifically, the invention relates to a method using oligonucleotides covalently linked to a solid support, such as beads, to isolate specific labeled nucleic acid sequences from complex mixtures. The methods disclosed allow quantitative comparisons of the amount of nucleic acid of defined sequence in a plurality of different samples of nucleic acid, e.g., from different cells or tissues or from genetic libraries. Nucleic acids from the samples are labeled in such a fashion that the signals can be distinguished and compared following hybridization to the oligonucleotides on the beads. According to the invention, the solid supports with the hybridized nucleic acid may be retrieved, and the target nucleic acid eluted and analyzed. Furthermore, the invention provides a method for tagging individual clones from a cDNA library such that they can be identified uniquely and retrieved by hybridization to specific beads.
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52 Claims
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1. A method for comparing relative amounts of specific nucleic acid molecules in at least two samples, comprising the steps of:
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(a) generating a target pool comprising a first and a second sample, wherein said first sample comprises nucleic acid molecules of a first source, and said first source nucleic acid molecules are linked to a first label, and wherein said second sample comprises nucleic acid molecules of a second source, and said second source nucleic acid molecules are linked to a second label;
(b) contacting said target pool with a plurality of solid supports each having attached thereto multiple capture oligonucleotides of a unique sequence under conditions which promote the formation of perfectly matched duplexes between said capture oligonucleotides and nucleic acid molecule complements within said target pool; and
(c) sorting the solid supports according to the relative amount of said first label and said second label;
wherein the unique capture oligonucleotides attached to each solid support comprise a stretch of from about 10 to about 40 nucleotides of random sequence, or a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from 7 to about 15 nucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 36, 37, 48, 50, 52)
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22. A method of normalizing a genetic library, comprising the steps of:
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(a) attaching unique oligonucleotide identifier tags to nucleic acid sequence inserts derived from a genetic library;
(b) hybridizing the inserts of step (a) with a nucleic acid sample derived from a source of interest under conditions that promote the formation of perfectly matched duplexes, wherein the nucleic acid sample is labeled with a first label;
(c) contacting the mixture of step (b) with solid supports having attached thereto the complements of the oligonucleotide identifier tags under conditions that promote the formation of perfectly matched duplexes between the oligonucleotide identifier tags and their respective complements in the presence of free oligonucleotide identifier tags labeled with a second label and corresponding in sequence to the oligonucleotide identifier tags of step (a);
(d) sorting solid supports according to the relative amount of said first label and said second label; and
(e) amplifying insert sequences present at lower abundance in order to match the abundance of insert sequences such that they are represented at substantially similar levels in the library. - View Dependent Claims (23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49)
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26. A method for comparing the relative amounts of specific nucleic acid molecules in at least two samples derived from a single nucleic acid source, wherein the relative abundance of one or more of said molecules changes during propagation in a host cell population, comprising the steps of:
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(a) introducing a nucleic acid sample from a starting nucleic acid source into a cell population;
(b) propagating the cell population;
(c) re-isolating the nucleic acid sample from the propagated cell population; and
(d) performing quantitative comparison of the relative amounts of at least one specific nucleic acid molecule in the propagated nucleic acid sample and the starting nucleic acid source.
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51. A nucleic acid comprising an oligonucleotide identifier tag, said tag comprising a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from 7 to about 15 nucleotides.
Specification