Combinatorial affinity selection
View Patent ↗In one aspect of the invention, methods for analyzing nucleic acid sample are provided. In a preferred embodiment, nucleic acids are selected using affinity matrices prior hybridization with a microarray.
1. A method for detecting and quantitating relative ratios of different nucleic acid sequence variants comprising:
selecting a first set of nucleic acids comprising a first target nucleic acid sequence from a sample comprising a mixture of nucleic acid sequences with a first nucleic acid affinity matrix comprising nucleic acid probes bound to at least one bead, said probes synthesized by combinatorial synthesis, wherein the first nucleic acid affinity matrix hybridizes, under stringent conditions comprising a salt concentration of 1 M and a temperature of at least 25° C., with the first set of nucleic acids;
selecting a second set of nucleic acids comprising a second target nucleic acid sequence from the sample with a second nucleic acid affinity matrix comprising nucleic acid probes bound to at least one bead, said probes synthesized by combinatorial synthesis, wherein the second nucleic acid affinity matrix hybridizes, under stringent conditions comprising a salt concentration of 1 M and a temperature of at least 25° C., with the second set of nucleic acids;
binding the sample with the affinity matrices;
washing the affinity matrices;
eluting the selected nucleic acids that are bound to the affinity matrices;
labeling the first set of selected nucleic acids with a first label;
labeling the second set of selected nucleic acids with a second label, wherein the second label is not the same as the first label;
hybridizing the first set of selected nucleic acids and the second set of selected nucleic acids to an array comprising probes which hybridize to the first and the second set of selected nucleic acids; and
detecting the first and second labels bound to the array, thereby quantitating the relative amounts of the first and second sets of selected nucleic acids.
2. The method according to claim 1 , wherein the first label is a first color and the second label is a second color, and wherein the first color and the second color are not the same.
3. The method according to claim 1 , wherein the labels are fluorescent labels.
4. The method according to claim 1 , wherein the first set of selected nucleic acids and the second set of nucleic acids are mixed together prior to hybridization to the array.
5. The method according to claim 1 , wherein the sample comprises transcript sequences.
6. The method according to claim 5 , wherein the first nucleic acid affinity matrix comprises probes complementary to a first transcript splice variant, wherein the second nucleic acid affinity matrix comprises probes complementary to a second transcript splice variant, and wherein the first and second transcript variants comprise different splice variant sequences.
7. The method according to claim 1 , wherein the first nucleic acid affinity matrix comprises probes complementary to a first sequence genotype, and wherein the second nucleic acid affinity matrix comprises probes complementary to a second sequence genotype whose sequence differs from the first sequence genotype by a single nucleotide polymorphism.
8. The method according to claim 1 , further comprising additional affinity matrices and corresponding different labels, allowing selection and relative quantitation of more than two different nucleic acid sequences.
9. The method according to claim 1 , wherein the beads are identified with coded oligonucleotides.
10. The method according to claim 1 , further comprising separation of the affinity matrices according to their magnetic properties.
11. The method according to claim 1 , wherein the beads comprise at least 100 different oligonucleotides.
12. The method according to claim 1 , wherein the beads comprise at least 1000 different oligonucleotides.
13. The method according to claim 1 , wherein the probes are synthesized on the beads.