Multiplex nucleic acid reactions
The invention is directed to a variety of multiplexing methods used to amplify and/or genotype a variety of samples simultaneously.
1. A multiplex method for determining whether a sample contains at least 100 different target sequences, comprising:
a) providing a sample which may contain at least 100 different single-stranded target sequences attached to a first solid support;
b) contacting said target sequences with a probe set comprising more than 100 different single-stranded probes, wherein each of said more than 100 different probes comprises:
i) a first universal priming site, wherein each of said more than 100 different probes has identical universal priming sites, and
ii) a target specific domain, such that different double-stranded hybridization complexes are formed, each of the different hybridization complexes comprising one of said more than 100 different single-stranded probes and one of the different single-stranded target sequences from the sample;
c) removing unhybridized probes;
d) contacting said probes of the hybridization complexes with a first enzyme and forming different modified probes;
e) contacting said modified probes with:
i) at least a first primer that hybridizes to said universal priming site;
ii) NTPs; and
iii) an extension enzyme;
wherein said different modified probes are amplified and forming different amplicons;
f) immobilizing said different amplicons to a second solid support, and
g) detecting said different amplicons immobilized to said second solid support, thereby determining whether the sample contains at least 100 different target sequences.
2. The method of claim 1 wherein each of said different target sequences comprises a detection position, each of said probes comprises an interrogation position, and said first enzyme modifies said probes if there is substantial complementarity between the bases at said interrogation position and said detection position.
3. The method of claim 1 , wherein said second solid support comprises an array.
4. The method of claim 3 , wherein said array comprises 1,000 probes per square cm.
5. The method of claim 1 or 3 , wherein each of said different probes in step b) further comprises a unique adapter sequence, whereby the modified probes formed in step d) comprise said unique adapter sequence and said amplicons formed in step e) comprise said unique adapter sequence.
6. The method of claim 5 , wherein said second solid support comprises capture probes that are specific to said unique adapter sequence of said amplicons.
7. The method of claim 1 or 3 , wherein each of said different probes further comprises an adapter sequence that differs from said target specific domain and that is used to uniquely identify the target sequence.
8. The method of claim 7 , wherein said second solid support comprises capture probes that are specific to said adapter sequence.
9. The method of claim 1 , wherein step (b) further comprises hybridizing said target sequences with a plurality of other probes and step (d) further comprises ligating said other probes to said probes.
10. The method of claim 1 or 9 , wherein said target sequences are covalently attached to said first solid support.
11. The method of claim 9 , wherein each of said other probes comprises a second universal priming site.
12. The method of claim 1 or 9 , wherein step (d) further comprises forming extended probes by extending said probes with a polymerase and NTPs having a biotin tag that allows removal of said extended probes.
13. The method of claim 9 , wherein each of said target sequences comprises a detection position, each of said probes comprises an interrogation position, and said first enzyme modifies said probes if there is substantial complementarity between the bases at said interrogation position and said detection position.
14. The method of claim 1 , 9 or 13 , wherein said first solid support comprises a plurality of beads.
15. The method of claim 14 , wherein said second solid support comprises a population of beads comprising capture probes that are specific to said amplicons.
16. The method of claim 15 , wherein said second solid support further comprises a substrate comprising wells, wherein each of said wells has a single bead from said population of beads.
17. The method of claim 1 , 3 , 9 or 11 , wherein said second solid support comprises a population of beads comprising capture probes that are specific to said amplicons.
18. The method of claim 17 , wherein said second solid support further comprises a substrate comprising wells, wherein each of said wells has a single bead from said population of beads.
19. The method of claim 9 , 11 or 13 , wherein step (d) further comprises extending said other probes with a polymerase and forming extended probes and ligating said extended probes to said probes.
20. The method of claim 9 , 11 or 13 , wherein step (d) further comprises extending said probes with a polymerase and forming extended probes, and ligating said extended probes to said other probes.
21. The method of claim 9 , 11 or 13 , wherein said second solid support comprises a population of beads comprising capture probes that are specific to said amplicons.
22. The method of claim 21 , wherein said second solid support further comprises a substrate comprising wells, wherein each of said wells has a single bead from said population of beads.