Multiplex nucleic acid reactions

• US 20030108900A1
• Filed: 07/12/2002
• Published: 06/12/2003
• Est. Priority Date: 07/12/2001
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Chee, Mark S., Oliphant, Arnold, Butler, Scott L., Stuelpnagel, John R.
• Granted Patent: US 7,582,420 B2
• 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. A method of detecting target sequences in a sample comprising:

a) providing a first solid support comprising at least a first and a second target sequence;

b) contacting said first and second target sequences with first and second probes, respectively, wherein each of said first and second probes comprise;

i) a first universal priming site;

ii) a target specific domain substantially complementary to at least a portion of said target sequence;

to form first and second hybridization complexes, respectively;

c) removing unhybridized probes;

d) contacting said first and second hybridization complexes with a first enzyme to form modified first and second probes, respectively;

e) contacting said modified first and second probes with;

i) at least a first primer that hybridizes to said universal priming site;

ii) NTPs; and

iii) an extension enzyme;

wherein said first and second modified probes are amplified to form first and second amplicons, respectively; and

f) detecting said amplicons.

2. A method according to claim 1 wherein each of said target sequences comprises a detection position, said probes each comprise an interrogation position, and said first enzyme modifies said probes if there is substantial complementarity between the bases at said interrogation position said detection position.

3. A method according to claim 1 or 2 wherein each of said probes further comprises a unique adapter sequence.

7. The method according to claim 1, wherein said unhybridized nucleic acids are removed by washing.

8. The method according to claim 1, said unhybridized nucleic acids are removed by enzymatic degradation.

9. The method according to claim 8, wherein said enzymatic degradation is by single stranded nucleases.

4. A method of detecting target sequences in a sample comprising:

a) providing a first solid support comprising at least a first and a second target sequence;

b) contacting said first and second target sequences with first and second probes, respectively, wherein each of said first and second probes comprise;

i) a first universal priming site;

ii) a target specific domain substantially complementary to at least a portion of said target sequence;

to form first and second hybridization complexes, respectively;

c) removing unhybridized probes;

d) contacting said first and second probes with;

i) at least a first universal primer that hybridizes to said universal priming site;

ii) NTPs; and

iii) an extension enzyme;

wherein said first and second probes are extended to form first and second modified probes, respectively;

e) contacting said first and second modified probes with;

i) at least third and fourth probes, respectively, wherein said modified first and second probes comprise a detection position, said third and fourth probes each comprise an interrogation position; and

ii) a second enzyme, wherein said second enzyme only modifies said third and

fourth probes if there is perfect complementarity between the bases at said interrogation position and said detection position, forming third and fourth modified probes; and

f) detecting said third and fourth modified probes.

5. A method comprising:

a) providing a plurality of target nucleic acid sequences each comprising from 3′

to 5′

a first, second and third target domain, said first target domain comprising a detection position, said second target domain being at least one nucleotide;

b) contacting said target nucleic acid sequences with sets of probes for each target sequence, each set comprising;

i) a first probe comprising from 5′

to 3′

;

1) a first domain comprising a first universal priming sequence; and

2) a second domain comprising;

A) a sequence substantially complementary to said first target domain of a target sequence; and

B) an interrogation position within the 3′

four terminal bases;

ii) a second probe comprising a first domain comprising a sequence substantially complementary to said third target domain of a target sequence;

to form a set of first hybridization complexes;

c) contacting said first hybridization complexes with;

i) an extension enzyme; and

ii) dNTPs;

under conditions whereby if the base at said interrogation positions is perfectly complementary with the bases at said detection positions, extension of said first probes occurs through said second target domains to form second hybridization complexes;

d) contacting said second hybridization complexes with a ligase to ligate the extended first probes to said second probes to form amplification templates.

6. A method according to claim 5 wherein said amplification templates are amplified to form amplicons and said amplicons are detected.

10. A multiplex reaction method comprising:

a. providing a sample comprising at least first and second targets;

b. hybridizing said first and second targets with first and second probes, respectively forming first and second hybridization complexes, respectively;

c. immobilizing said first and second hybridization complexes;

d. washing to remove unhybridized nucleic acids;

e. contacting said first and second hybridization complexes with an enzyme, whereby said first and second probes are modified forming modified first and second probes, respectively, whereby said modified first and second probes are modified to contain first and second interrogation nucleotides that are complementary to first and second detection nucleotides in said first and second targets, respectively;

f. contacting said modified first and second probes with;

i. first and second allele specific primers, respectively, whereby said first and second allele specific primers hybridize to said modified first and second probes, respectively, 5′

to said first and second interrogation nucleotides;

ii. dNTPs

iii. polymerase,

whereby said first and second allele specific primers are modified when a target domain of said allele specific primers is perfectly complementary to said modified target probes to form modified first and second allele specific probes;

g. amplifying said modified first and second allele specific probes to form first and second amplicons; and

h. detecting said first and second amplicons.

11. A method comprising:

a) providing a plurality of target nucleic acid sequences each comprising from 3′

to 5′

a first, second and third target domain, said first target domain comprising a detection position, said second target domain being at least one nucleotide;

b) contacting said target nucleic acid sequences with sets of probes for each target sequence, each set comprising;

i) a first probe comprising from 5′

to 3′

;

1) a first domain comprising a first universal priming sequence; and

2) a second domain comprising;

A) a sequence substantially complementary to said first target domain of a target sequence; and

B) an interrogation position one, two or three nucleotides from the 3′

terminal base of said second domain;

ii) a second probe comprising a first domain comprising a sequence substantially complementary to said third target domain of a target sequence;

to form a set of first hybridization complexes;

c) contacting said first hybridization complexes with;

i) at least a first universal primer that hybridize to said first universal priming sequence;

ii) an extension enzyme; and

iii) dNTPs;

under conditions whereby if the base at said interrogation positions are perfectly complementary with the bases at said detection positions, extension of said first probes occurs through said second target domains to form second hybridization complexes;

d) contacting said second hybridization complexes with a ligase to ligate the extended first probes to said second probes to form amplification templates.

12. The method according to claim 11, wherein said first probe further comprises said first domain and said second domain, a third domain comprising a unique adapter sequence.

13. The method according to claim 11 or 12, further comprising contacting said amplification templates with universal primers, an extension enzyme and dNTPs, whereby said amplification templates are amplified forming amplicons.

14. The method according to claim 13, further comprising detecting said amplicons.