Multiplexed methylation detection methods

• US 20030170684A1
• Filed: 12/03/2002
• Published: 09/11/2003
• Est. Priority Date: 02/07/2000
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Fan, Jian-Bing
• Granted Patent: US 7,611,869 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• 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 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention is directed to sensitive and accurate multiplexed assays for target analyte detection and detection of methylation in nucleic acid samples.

Claims

1. A method for multiplex detection of methylation of target nucleic acids comprising:

a. providing a first population of target nucleic acids labeled with a purification tag;

b. cleaving said first population of target nucleic acids with an enzyme, whereby said enzyme discriminately cleaves at methylated target sequences forming a second population of cleaved target sequences;

c. immobilizing said first and second populations by said purification tag; and

d. detecting the presence of said first population comprising non-cleaved target nucleic acid whereby the presence of said first population comprising non-cleaved target nucleic acid indicates the presence of methylated target nucleic acids.

2. The method according to claim 1, wherein said purification tag comprises biotin and said first and second labeled target nucleic acids are immobilized with streptavidin.

3. The method according to claim 1, wherein said enzyme is selected from the group consisting of whatever they are

4. The method according to claim 1, wherein said detecting comprises:

e. contacting said immobilized first and second populations with a composition comprising a plurality of target probes forming a plurality of hybridization complexes, said probes comprising;

i) a first region complementary to a first region of a target nucleic acid; and

ii) a second region comprising a detection sequence complementary to a potentially methylated nucleotide; and

f. detecting the presence of said probe as an indication of the presence of methylated target nucleic acid.

5. The method according to claim 4, wherein said probes further comprise at least a first universal priming sequence and f) comprises:

i) contacting said hybridization complexes with a composition comprising;

a) at least first universal primers;

b) dNTPs; and

c) polymerase,

whereby said probes are amplified forming a plurality of amplicons; and

ii) detecting said amplicons as an indication of the presence of methylated target nucleic acid.

8. The method according to claim 5 or 7, wherein said probes are amplified by a method selected from the group consisting of oligonucleotide ligation assay (OLA), polymerase chain reaction (PCR) and rolling circle amplification (RCA).

9. The method according to claim 8, wherein said probes are amplified by oligonucleotide ligation assay (OLA).

10. The method according to claim 8, wherein said probes are amplified by polymerase chain reaction (PCR).

11. The method according to claim 8, wherein said probes are amplified by rolling circle amplification (RCA).

12. The method according to claim 5 or 7, wherein said amplicons are detected by hybridizing said amplicons to an array.

13. The method according to claim 12, wherein said array is selected from the group consisting of an ordered array, a liquid array and a random array.

14. The method according to claim 5 or 7, wherein said amplicons are detected by mass spectrometry.

15. The method according to claims 5 or 7, wherein said amplicons are detected by capillary electrophoresis.

6. A method of detecting methylation comprising:

a. contacting a sample of target nucleic acids with bisulfite, whereby non-methylated cytosine is converted to uracil, forming a first population of treated target nucleic acids, and methylated cytosine is not converted to uracil, forming a second population of treated target nucleic acids;

b. contacting said first and second populations of treated target nucleic acids with a first probe that hybridizes with a first target in said first population of target nucleic acid and a second probe that hybridizes with a target in said second population of target nucleic acid, forming first and second hybridization complexes, respectively;

c. contacting said first and second hybridization complexes with an enzyme that modifies said first and second probes forming first and second modified probes;

d. detecting said first and second modified probes to determine the presence of methylation in said target nucleic acid.

7. A method according to claim 6, wherein said detecting comprises:

i) contacting said hybridization complexes with a composition comprising;

a) at least first universal primers;

b) dNTPs; and

c) polymerase,

whereby said probes are amplified forming a plurality of amplicons; and

ii) detecting said amplicons as an indication of the presence of methylated target nucleic acid.