MULTIPLEX NUCLEIC ACID REACTIONS

• US 20080242555A1
• Filed: 02/15/2008
• Published: 10/02/2008
• Est. Priority Date: 02/07/2000
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Fan, Jian-Bing, Kuhn, Kenneth M., Chee, Mark S., Oliphant, Arnold, Butler, Scott L., Shen, Min-Jui Richard, Stuelpnagel, John R.
• Granted Patent: US 8,003,354 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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

1-11. -11. (canceled)

12. A multiplex genotyping method, comprising (a) providing a first solid support comprising a plurality of at least 100 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 different probe sets,

wherein each probe set comprises an allele specific probe and a locus specific probe,

wherein said allele specific probe comprises a first universal priming sequence and a target specific portion that discriminates between single nucleotide polymorphism alleles of said plurality of different target nucleic acids, 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 sequences to form a plurality of hybridization complexes;

(d) extending said allele specific probes of said hybridization complexes in an allele specific manner to form an extended probes;

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

(f) amplifying said ligated probes to form amplicons comprising said adapter sequences;

(g) hybridizing said amplicons to capture probes on a second solid support, wherein said capture probes are specific for individual adapter sequences of said amplicons; and

(h) detecting said amplicons that are hybridized to said capture probes to discriminate between single nucleotide polymorphism alleles of said plurality of at least 100 different target nucleic acids.

13. The method of claim 12, wherein the terminal base of said allele specific probe corresponds to said SNP allele.

14. The method of claim 12, wherein each probe set further comprises a second allele specific probe, wherein said allele specific probe comprises a match for a SNP at a detection position and said second allele specific probe comprises a mismatch for said SNP at said detection position.

15. The method of claim 12, wherein said amplifying comprises PCR using a single pair of universal primers that are complementary to said first universal priming sequence and said second universal priming sequence.

16. The method of claim 12, wherein said amplifying comprises PCR using primers having fluorescent labels.

17. The method of claim 12, wherein said first solid support comprises a plurality of beads.

18. The method of claim 17, wherein said beads are attached to said target sequences using a purification tag.

20. The method of claim 12, wherein said second solid support comprises an array of said capture probes.

21. The method of claim 12, wherein said array comprises 1,000 probes per square cm.

22. The method of claim 12, wherein step (d) comprises extending said allele specific probe of said hybridization complex to add a base to basepair with said SNP allele.

19. The method of claim 19, wherein said beads are attached to said target sequences by a covalent attachment.