MULTIPLEX NUCLEIC ACID REACTIONS

• US 20100015626A1
• Filed: 07/21/2009
• Published: 01/21/2010
• Est. Priority Date: 02/07/2000
• Status: Abandoned Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Fan, Jian-Bing, Chee, Mark S., Oliphant, Arnold, Butler, Scott L., Shen, Min-Jui Richard, Stuelpnagel, John R.
• Application Number:
  US12/507,022
• Publication Number:
  US 20100015626A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention is directed to a variety of multiplexing methods used to amplify and/or genotype a variety of samples simultaneously.

Claims

1-14. -14. (canceled)

15. A method for detecting 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, two, three or four nucleotides from the 3′

terminal base of the second target domain, and the second target domain is at least one nucleotide, comprising the steps of;

(a) providing a sample having different target nucleic acid sequences of interest;

(b) contacting the sample with dNTPs and primers that hybridize to the first target domains, and extending the primers to obtain first extension products;

(c) contacting the first extension products 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 universal 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 universal priming sequence,

wherein at least one probe contains a distinct adapter sequence not native to the target sequence of interest;

(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 universal primers to produce amplicons;

(g) immobilizing the amplicons on solid phase capture probes that are specific to individual adapter sequences; and

(h) detecting the presence of different immobilized amplicons at the capture probes;

thereby indicating the presence of the different target sequences of interest in the sample.

16. The method of claim 15, wherein the first extension product is purified from the sample.

17. The method of claim 16, wherein the dNTPs are labeled, and the first extension product is purified by means of the label.

18. The method of claim 16, wherein the primer is tagged with a label, and the first extension product is purified by means of the label.

19. The method of claim 18, wherein the primer is tagged with biotin or streptavidin.

20. The method of claim 18, wherein prior to step (c), the first extension products are immobilized to a solid support.

21. The method of claim 15, wherein the nucleic acids in step (a) comprise single nucleotide polymorphism alleles.

22. The method of claim 15, wherein said target sequences comprise single nucleotide polymorphism alleles and said probe sets comprise allele-specific probes that discriminate between said alleles.

23. The method of claim 15, wherein a set of probes for each of at least 100 different target sequences of interest are contacted in step (b), thereby indicating of the presence of the at least 100 different target sequences of interest in the sample.

24. The method of claim 15, wherein a set of probes for each of at least 200 different target sequences of interest are contacted in step (b), thereby indicating of the presence of the at least 200 different target sequences of interest in the sample.

25. The method of claim 15, wherein the detection position is one nucleotide from the 3′

-terminal base of the second domain.

26. The method of claim 15, wherein the detection position is two nucleotides from the 3′

-terminal base of the second domain.

27. The method of claim 15, wherein the second target domain is 1 nucleotide in length.

28. The method of claim 15, wherein the second target domain is from 2 to 5 nucleotides in length.

29. The method of claim 15, wherein each set of probes further comprises a third probe comprising from 5′

to 3′

;

the third universal priming sequence and a sequence that is substantially complementary to the first target domain and that has an interrogation position corresponding to the detection position, wherein the interrogation position is not complementary to the base at the detection position, whereby the third probe is not extended in step (c).

30. The method of claim 15, wherein each of said probe sets comprises a universal priming sequence that is the same for other probe sets.

31. The method of claim 15, wherein a universal primer is detectably labeled.

32. The method of claim 15, wherein the first universal primers in step (e) comprise a detectable label.

33. The method of claim 15, wherein the first probe comprises the adapter sequence, whereby an amplicon comprises from 5′

to 3′

;

the first universal primer, the adapter sequence, the first target domain, the second target domain, the third target domain, and the second universal primer.

34. The method of claim 15, wherein the second probe comprises the adapter sequence, whereby an amplicon comprises from 5′

to 3′

;

the first universal primer, the first target domain, the second target domain, the third target domain, the adapter sequence, and the second universal primer.

35. The method of claim 15, wherein step (e) comprises performing PCR using a single pair of universal primers that are complementary to said first universal priming sequence and said second universal priming sequence.

36. The method of claim 15, wherein the hybridization complexes are washed while immobilized on the solid support under conditions sufficient to remove non-hybridized nucleic acids.

37. The method of claim 15, wherein the amplification templates are immobilized on the solid support and are washed under conditions sufficient to remove non-hybridized nucleic acids.

38. The method of claim 15, wherein the second probe comprises a sequence that is complementary to the third target domain.

39. The method of claim 38, wherein ligation in step (d) occurs only when the 3′

-end of the extended first probe and the 5′

-end of the second probe are perfectly complementary to the target sequence.

40. The method of claim 15, wherein the second probe comprises an interrogation position.

41. The method of claim 40, wherein the interrogation position is at the 5′

end of the second probe.

42. The method of claim 15, wherein no significant ligation occurs between the extended first probe and the second probe unless the interrogation position of the second probe is perfectly complementary to the target sequence.

43. The method of claim 15, wherein step (a) comprises providing double-stranded sample and denaturing the sample into separate strands, and wherein the primers in step (b) comprise primers that hybridize to one strand and primers that hybridize to the other strand.