Methods of copying the methylation pattern of DNA during isothermal amplification and microarrays

• US 7,449,297 B2
• Filed: 04/11/2006
• Issued: 11/11/2008
• Est. Priority Date: 04/14/2005
• Status: Expired due to Fees

• Current Assignee: Euclid Diagnostics Lcc, Euclid Diagnostics LLC
• Original Assignee: Euclid Diagnostics LLC
• Inventors: Freije, Wadiha, Brikun, Igor
• Primary Examiner(s): Strzelecka; Teresa E
• Application Number:
  US11/402,681
• Publication Number:
  US 20060257905A1
• Time in Patent Office: 945 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

A method for copying the methylation patterns of molecules of genomic DNA (MGD) during isothermal amplification of the MGD comprising obtaining MGD, copying the methylation patterns of the MGD using a DNA methylation-maintenance enzyme, while isothermally amplifying the MGD using a DNA polymerase with strand displacement activity, under conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase; a method for copying the methylation patterns in double-stranded DNA molecules during isothermal amplification of the DNA molecules comprising obtaining DNA molecules, contacting the DNA molecules with transposable elements and an enzyme, which can randomly insert the transposable elements into the DNA molecules, copying the methylation patterns of the DNA molecules using a DNA methylation-maintenance enzyme, while isothermally amplifying the DNA molecules using a DNA polymerase with strand displacement activity, under conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase; a buffer comprising a divalent ion, deoxynucleotide triphosphates (dNTPs), primers, and S-adenosyl-methionine (SAM); and a composition comprising a DNA methylation-maintenance enzyme, a DNA polymerase with strand displacement activity, and the buffer.

Claims

1. A method for copying the methylation patterns of molecules of genomic DNA (MGD) during isothermal amplification of the MGD, which method comprises:

(i) obtaining MGD,

(ii) copying the methylation patterns of the MGD using a DNA methylation-maintenance enzyme, while isothermally amplifying the MGD using a DNA polymerase with strand displacement activity, under conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase with strand displacement activity, wherein the DNA methylation-maintenance enzyme is DNA methyltransferase 1 (DNMT-1) and wherein the DNMT- 1 is optionally modified to reduce or eliminate de novo methylation activity, and

(iii) optionally purifying the MGD, whereupon the MGD are amplified and their methylation patterns are copied.

2. The method of claim 1, wherein the DNMT-1 is from a eukaryote.

3. The method of claim 2, wherein the eukaryote is a human.

4. The method of claim 1, wherein the MGD are completely or partially double-stranded, and wherein the method further comprises contacting the MGD with transposable elements and an enzyme to randomly insert the transposable elements into the MGD to provide MGD comprising the randomly inserted transposable elements.

5. The method of claim 1, wherein the conditions that simultaneously promote activity of the DNA methylation-maintenance enzyme and the DNA polymerase with strand displacement activity comprise the presence of a buffer comprising a divalent ion, deoxynucleotide triphosphates (dNTPs), primers, and S-adenosyl-methionine (SAM).

6. The method of claim 1, wherein step (ii) further comprises:

(i′

) mixing the MGD and primers, wherein either of (a) the MGD or (b) the MGD and the primers are optionally denatured before or after mixing,

(ii′

) incubating the mixture of (i′

) at a temperature from about 0 °

C. to about 75°

C. for a sufficient amount of time to allow annealing of the primers to the MGD,

(iii′

) equilibrating the mixture of (ii′

) to a temperature for methyltransferase activity,

(iv′

) adding a buffer, which comprises a divalent ion, dNTPs, primers, and SAM, and which is already equilibrated to the temperature of (iii′

), and simultaneously with or sequentially to adding the buffer, adding the DNA methylation-maintenance enzyme and the DNA polymerase with strand displacement activity to the mixture of (iii′

), and

(v′

) incubating the mixture of (iv′

) at a temperature for methyltransferase activity to allow methylation-coupled amplification of the MGD until a desired amount is obtained of the MGD.

7. The method of claim 1, wherein the MGD are obtained from circulating DNA.

8. The method of claim 1, wherein the MGD comprise a mixture of fragments of strands of different length.

9. The method of claim 8, which further comprises contacting the MGD with an enzyme to provide MGD fragments with blunt ends.

10. The method of claim 9, wherein the blunt ends are generated by extending shorter strands of the fragments, by shortening longer strands of the fragments, or a combination thereof, optionally in the presence of a polynucleotide kinase.

11. The method of claim 9, which method further comprises contacting the MGD with an enzyme that ligates double-stranded nucleic acids under reaction conditions that promote ligation, optionally in the presence of a polynucleotide kinase.

12. The method of claim 10, which method further comprises contacting the MGD with an enzyme that ligates double-stranded nucleic acids under reaction conditions that promote ligation, optionally in the presence of a polynucleotide kinase.

13. A composition for methylation-coupled amplification of DNA comprising a DNMT-1, a DNA polymerase with strand displacement activity, and a buffer comprising a divalent ion, dNTPs, primers, and SAM, wherein the buffer is suitable for simultaneously promoting the activity of a DNA methylation-maintenance enzyme and a DNA polymerase with strand displacement activity and wherein the composition lacks a divalent cation chelator.