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 method for amplifying different target nucleic acid sequences of interest in a sample,
each sequence comprising, from 3′ to 5′: contiguous first, second, and third target domains, wherein
the first target domain has a detection position one nucleotide from the 3′ terminal base of the second target domain, and
the second target domain is at least 100 nucleotides in length,
comprising the steps of:
(a) providing a sample having different target nucleic acid sequences of interest;
(b) contacting the sample with a set of probes for each of the different target nucleic acid sequences of interest to form hybridization complexes, each set comprising:
a first probe comprising, from 5′ to 3′: a first priming sequence and a sequence that is substantially complementary to the first target domain and that has an interrogation position suitable for basepairing with the detection position; and
a second probe comprising 5′ to 3′: a sequence substantially complementary to the third target domain, and a second priming sequence,
wherein at least one probe contains a distinct adapter sequence not native to the target sequence of interest;
(c) immobilizing the hybridization complexes on a solid support;
(d) contacting the hybridization complexes with an extension enzyme and dNTPs, wherein for each hybridization complex,
if the base at the interrogation position is perfectly complementary to the base at the detection position, then the first probe is extended along the second target domain;
(e) ligating the extended first probes to second probes to form amplification templates;
(f) amplifying the amplification templates with first and second primers to produce amplicons; and
(g) immobilizing the amplicons on solid phase capture probes;
thereby amplifying the different target sequences of interest in the sample.
2. The method of claim 1 , wherein a set of probes for each of at least 100 different target sequences of interest are contacted in step (b), thereby amplifying at least 100 different target sequences of interest in the sample.
3. The method of claim 1 , wherein a set of probes for each of at least 200 different target sequences of interest are contacted in step (b), thereby amplifying at least 200 different target sequences of interest in the sample.
4. The method of claim 1 , wherein a set of probes for each of at least 500 different target sequences of interest are contacted in step (b), thereby amplifying at least 500 different target sequences of interest in the sample.
5. The method of claim 1 , wherein a set of probes for each of at least 1000 different target sequences of interest are contacted in step (b), thereby amplifying at least 1000 different target sequences of interest in the sample.
6. The method of claim 1 , wherein the solid support comprises a plurality of beads.
7. The method of claim 1 , wherein the solid support comprises a first binding partner capable of binding to a second binding partner, and the nucleic acids are modified with the second binding partner.
8. The method of claim 7 , wherein the first binding partner is streptavidin.
9. The method of claim 1 , wherein the primers are universal primers.
10. The method of claim 1 , wherein the amplicons are immobilized on solid-phase capture probes that are specific to individual adapter sequences.
11. The method of claim 1 , further comprising the step of
(h) detecting the different immobilized amplicons at the capture probes by sequencing.
12. The method of claim 11 , wherein the sequencing in step (h) is pyrosequencing.
13. A method for amplifying different target nucleic acid sequences of interest, comprising performing steps of the method of claim 1 on a variety of samples.
14. The method of claim 13 , wherein the steps are performed on a variety of samples simultaneously.
15. The method of claim 1 , wherein unreacted probes in step (b) are removed by filtration.
16. The method of claim 1 , wherein each of the first primers comprises an adapter sequence that serves as an identifier for target sequence.
17. The method of claim 1 , wherein each of the second primers comprises an adapter sequence that serves as a unique identifier for the target sequence.
18. The method of claim 1 , wherein one of the primers is detectably labeled.
19. The method of claim 18 , wherein the detectable label is fluorescent.
20. The method of claim 18 , wherein the detectable label is biotin.