Detection of nucleic acid reactions on bead arrays
The present invention is directed to methods and compositions for the use of microsphere arrays to detect and quantify a number of nucleic acid reactions. The invention finds use in genotyping, i.e. the determination of the sequence of nucleic acids, particularly alterations such as nucleotide substitutions (mismatches) and single nucleotide polymorphisms (SNPs). Similarly, the invention finds use in the detection and quantification of a nucleic acid target using a variety of amplification techniques, including both signal amplification and target amplification. The methods and compositions of the invention can be used in nucleic acid sequencing reactions as well. All applications can include the use of adapter sequences to allow for universal arrays.
1. A method of detecting a plurality of target nucleic acid sequences, comprising:
a) providing one or more first solid supports comprising a plurality of target sequences that are immobilized to said first solid supports, wherein said first solid supports comprise a plurality of beads;
b) hybridizing a plurality of different first primers to first portions of said plurality of target sequences, wherein each of said different first primers comprises an adapter sequence exogenous to said target sequences;
c) hybridizing a plurality of different second primers to second portions of said plurality of target sequences, thereby forming a plurality of hybridization complexes;
d) extending said first or said second primers, and ligating said first and second primers together to form a plurality of different modified primers;
e) hybridizing said adapter sequences of said modified primers or their complements to an array of capture probes attached to discrete sites on a second solid support;
f) modifying said capture probes by polymerase extension, and
g) detecting said modified capture probes, thereby detecting said plurality of target nucleic acid sequences.
2. The method of claim 1 , wherein after said extending of said first or second primers, hybridizing said plurality of different modified primers with a plurality of amplifier probes complementary to said plurality of first and second primers and amplifying said different modified primers, thereby forming amplified products comprising said adapter sequences or their complements.
3. The method of claim 2 , further comprising hybridizing said adapter sequences of said amplified products or their complements to said array of capture probes.
4. The method of claim 2 , comprising detecting a label attached to said amplified products.
5. The method of claim 2 , wherein said amplifying comprises a polymerase chain reaction.
6. The method of claim 2 , further comprising extending said amplifier probes with a polymerase and NTPs.
7. The method of claim 2 , wherein said amplifier probes further comprise a label.
8. The method of claim 7 , wherein said label comprises a biotin tag that allows isolation of extended amplifier probes.
9. The method of claim 2 , further comprising a wash step prior to amplifying said different modified primers.
10. The method of claim 2 , further comprising extending said amplifier probes with a polymerase and dNTPs.
11. The method of claim 1 , further comprising identifying a nucleotide at a detection position for each of said target nucleic acid sequences, wherein a primer of said plurality of first primers or said plurality of second primers is complementary to said detection position.
12. The method of claim 1 , wherein said target sequences are attached to said first solid supports.
13. The method of claim 1 , wherein said array comprises a population of beads comprising said capture probes.
14. The method of claim 13 , wherein said beads are associated with individual sites of a substrate.
15. The method of claim 14 , wherein each of said sites is configured to have a single associated bead.
16. The method of claim 14 , wherein said substrate comprises a fiber optic bundle.
17. The method of claim 1 , wherein said array is made by a method selected from the group consisting of a spotting technique, photolithographic technique, and printing technique.
18. The method of claim 1 , comprising detecting a label attached to said modified primers.
19. The method of claim 1 , comprising detecting a fluorescent label.
20. The method of claim 1 , wherein said plurality of target sequences are immobilized by covalent attachment to said first solid supports.
21. The method of claim 1 , wherein said capture probes are covalently attached to said second solid support.
22. The method of claim 1 , wherein each of said target sequences comprises a locus having a single nucleotide polymorphism (SNP) allele.
23. The method of claim 22 , wherein said plurality of said first primers comprise allele specific primers and said plurality of second primers comprise locus specific primers.
24. The method of claims 23 , wherein the terminal base of said allele specific primers correspond to said SNP allele.
25. The method of claim 23 , further comprising a plurality of second allele specific primers, wherein said plurality of allele specific primer comprises a match for said SNP and said plurality of second allele specific primers comprises a mismatch for said SNP at said detection position.
26. The method of claims 23 , wherein step (d) comprises extending said allele specific probe of said hybridization complexes to add a base to basepair with said SNP.
27. The method of claim 23 , wherein said detecting of step (g) indicates a SNP clinically related to a phenotypic variant.
28. The method of claim 23 , wherein said detecting of step (g) determines the genotype of a target sequence that is an indicator of a disease.
29. The method of claim 1 , wherein said detecting of step (g) indicates the amount of a target sequence present in said plurality of nucleic acid sequences.
30. The method of claim 1 , wherein said detecting of step (g) determines the presence of a target sequence that is an indicator of a disease.
31. The method of claim 1 , wherein said first and second primers are separated by one or more bases.