DIFFERENTIAL ENZYMATIC FRAGMENTATION

• US 20100240064A1
• Filed: 06/01/2010
• Published: 09/23/2010
• Est. Priority Date: 10/21/2003
• Status: Active Grant

• Current Assignee: Orion Genomics LLC
• Original Assignee: Orion Genomics LLC
• Inventors: Lakey, Nathan D., Jeddeloh, Jeffrey A.
• Granted Patent: US 8,163,485 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2545/101 with an internal standard/control

Abstract

The present invention provides methods for detecting the presence of methylation at a locus within a population of nucleic acids.

Claims

1. A method of detecting the presence of methylation at a locus within a population of nucleic acids, the method comprising

(a) dividing the population of nucleic acids into at least two portions,

(b) contacting a first portion with a methylation-sensitive restriction enzyme to obtain a population comprising fragmented unmethylated copies of the locus and intact methylated copies of the locus;

(c) quantifying the intact copies of the locus in the first portion;

(d) contacting a second portion with a methylation-dependent restriction enzyme to obtain a population comprising fragmented methylated copies of the locus and intact unmethylated copies of the locus;

(e) quantifying the intact copies of the locus in the second portion; and

(f) determining the presence of methylation at the locus by comparing the number of intact copies of the locus in the first portion and number of intact copies of the locus in the second portion.

2. The method of claim 1, further comprising

quantifying a third portion of the nucleic acids, thereby amplifying the total intact copies of the locus in the population;

and

comparing the number of total intact copies of the locus to the number of intact copies of the locus in the first portion and/or intact copies of the locus in the second portion.

3. The method of claim 1, further comprising contacting a third portion of the nucleic acids with a methylation-dependent restriction enzyme and a methylation-sensitive restriction enzyme;

quantifying copies of the intact locus in the third portion; and

determining the presence of methylation at the locus by comparing the number of the intact copies of the locus in the third portion to the number of intact copies of the locus in the first portion and/or intact unmethylated copies of the locus in the second portion.

4. The method of claim 1, wherein the second portion is also contacted with the methylation-sensitive restriction enzyme prior to the step of quantifying the intact copies of the locus in the second portion.

5. The method of claim 1, wherein the first portion is also contacted with the methylation-dependent restriction enzyme prior to the step of quantifying the intact copies of the locus in the first portion.

6. The method of claim 2, further comprising contacting a fourth portion of the nucleic acids with a methylation-dependent restriction enzyme and a methylation-sensitive restriction enzyme;

quantifying intact copies of the locus in the fourth portion; and

determining the presence of methylation at the locus by comparing the number of intact copies of the locus in the fourth portion to the number of total intact copies of the locus in the third portion and/or intact copies of the locus in the first portion and/or intact copies of the locus in the second portion.

7. The method of claim 2, wherein the second portion is also contacted with the methylation-sensitive restriction enzyme prior to the step of quantifying the intact copies of the locus in the second portion.

8. The method of claim 2, wherein the first portion is also contacted with the methylation-dependent restriction enzyme prior to the step of quantifying the intact copies of the locus in the first portion.

9. The method of claim 6, further comprising identification of mutations within the locus.

10. The method of claim 9, wherein a fifth portion of the nucleic acids is contacted with a methylation-insensitive restriction enzyme;

quantifying intact copies of the locus in the fifth portion to obtain a population of nucleic acids comprising a mutation at the locus; and

determining the presence of a mutation at the locus by comparing the number of the intact copies of the locus in the fifth portion to the number of intact copies of the locus in the fourth portion and/or total intact copies of the locus in the third portion and/or intact copies of the locus in the first portion and/or intact copies of the locus in the second portion.

11. The method of claim 1, wherein the quantifying steps comprise quantitative amplification.

12. The method of claim 11, wherein the nucleic acids are contacted with an agent that modifies unmethylated cytosines before the amplification step;

and the intact copies of the locus are amplified with a pair of oligonucleotide primers comprising at least one primer that distinguishes between modified methylated and unmethylated DNA.

13. The method of claim 1, wherein the methylation-dependent restriction enzyme is McrBC.

14. A kit, comprising:

a methylation-sensitive restriction enzyme; and

a methylation-dependent restriction enzyme.