Multiplex nucleic acid reactions

• US 8,003,354 B2
• Filed: 02/15/2008
• Issued: 08/23/2011
• Est. Priority Date: 02/07/2000
• Status: Expired due to Term

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Shen, Min-Jui Richard, Stuelpnagel, John R., Fan, Jian-Bing, Kuhn, Kenneth M., Chee, Mark S., Oliphant, Arnold, Butler, Scott L.
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US12/032,581
• Publication Number:
  US 20080242555A1
• Time in Patent Office: 1,285 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/287.2, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays

Abstract

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

Claims

1. A multiplex detection method, comprising

(a) providing a first solid support comprising a plurality of different target nucleic acids,

wherein each of said target nucleic acids comprises a locus having a single nucleotide polymorphism (SNP) allele;

(b) providing a plurality of at least 100 different probe sets, wherein each of said probe sets comprises a first allele specific probe and a locus specific probe,

wherein said first allele specific probe comprises a first universal priming sequence and a target specific portion that is complementary to a specific target nucleic acid of said plurality of different target nucleic acids and is capable of identifying the single nucleotide polymorphism allele of said specific target nucleic acid, and

wherein said locus specific probe comprises a second universal priming sequence, an adapter sequence and a target specific portion that is complementary to at least a portion of said locus;

(c) hybridizing said probe sets to said target nucleic acids and forming a plurality of hybridization complexes;

(d) extending the first allele specific probe of each of said hybridization complexes in an allele specific manner under sufficient conditions so that only the first allele specific probe of each of said hybridization complexes that is perfectly complementary to the SNP allele of a target nucleic acid of said plurality of different target nucleic acids is extended, and forming extended probes;

(e) ligating said extended probes to said locus specific probes and forming ligated probes;

(f) amplifying said ligated probes and forming amplicons, each of said amplicons comprising said adapter sequence;

(g) hybridizing said amplicons to capture probes on a second solid support having at least 100 different said capture probes,

wherein each of said capture probes is complementary to said adapter sequence; and

(h) detecting said amplicons that are hybridized to said capture probes;

thereby detecting said single nucleotide polymorphism allele of each of said plurality of different target nucleic acids.

2. The method of claim 1, wherein each of the probe sets further comprises a second allele specific probe, wherein said first allele specific probe comprises a base that is complementary to said SNP allele and said second allele specific probe comprises a base that is noncomplementary to said SNP allele.

3. The method of claim 1, wherein said first solid support comprises a plurality of beads.

4. The method of claim 1, 2 or 3, wherein the terminal base of said first allele specific probe hybridizes to said SNP allele.

5. The method of claim 1, 2 or 3, wherein said amplifying step comprises PCR using a single pair of universal primers that are complementary to said first universal priming sequence and said second universal priming sequence.

6. The method of claim 1, 2 or 3, wherein said amplifying step comprises PCR using primers having fluorescent labels.

7. The method of claim 1, 2 or 3, wherein said beads are attached to said target nucleic acids using a purification tag.

8. The method of claim 3, wherein said beads are attached to said target nucleic acids by a covalent attachment.

9. The method of claim 1, 2 or 3, wherein said second solid support comprises an array of said capture probes.

10. The method of claim 9, wherein said array comprises 1,000 said capture probes per square cm.