Compositions and methods for performing methylation detection assays

Patent metadata

Field	Value
Publication number	US-11118230-B2
Application number	US-201816013240-A
Country	US
Kind code	B2
Filing date	Jun 20, 2018
Priority date	Dec 12, 2014
Publication date	Sep 14, 2021
Grant date	Sep 14, 2021

Abstract

Provided herein is technology relating to performing methylation assays. In particular, the technology relates to internal controls for methylation assays.

First claim

We claim:

1. A composition, comprising:
a) a complex of a bisulfite-treated ZDHHC1 DNA comprising SEQ ID NO:33 or its complement, and at least one oligonucleotide complementary to SEQ ID NO:33 or its complement, wherein said oligonucleotide is specifically hybridized to a site in said bisulfite-treated ZDHHC1 DNA within SEQ ID NO:33 or its complement, wherein the at least one oligonucleotide complementary to SEQ ID NO:33 or its complement is a detection probe oligonucleotide; and
b) complexes of two to seven marker DNAs selected from the group consisting of vimentin, septin 9, NDRG4, BMP3, VAV3, SFMBT2, and TFPI2a, and oligonucleotides specifically hybridized to the marker DNAs;
wherein the complexes are in one or more reaction mixtures that each comprise one or more of a FRET cassette and a FEN-1 endonuclease.

2. The composition of claim 1 , wherein said oligonucleotides specifically hybridized to the two to seven marker DNAs are selected from one or more of a capture oligonucleotide, a pair of nucleic acid primers, a probe oligonucleotide, and an invasive oligonucleotide.

3. The composition of claim 1 , in a reaction mixture with one or more nucleic acids selected from the group consisting of: oligonucleotides of SEQ ID NOS:8-10, 52, 56-60, and 63-65, a strand of DNA comprising the nucleotide sequence of SEQ ID NO:54, a strand of DNA comprising the nucleotide sequence of SEQ ID NO:61, and a strand of DNA comprising the nucleotide sequence of SEQ ID NO: 68.

4. The composition of claim 1 , wherein said detection probe oligonucleotide comprises a reporter molecule.

5. The composition of claim 4 , where said reporter molecule comprises a fluorophore.

6. The composition of claim 1 , wherein said detection probe oligonucleotide comprises a flap sequence.

7. The composition of claim 1 , further comprising a thermostable DNA polymerase.

8. The composition of claim 1 , wherein the two to seven marker DNAs are bisulfate-treated genomic DNAs.

9. The composition of claim 1 , wherein a reaction mixture comprising the complex of a bisulfate-treated ZDHHC1 DNA comprising SEQ ID NO:33 or its complement and the at least one oligonucleotide complementary to SEQ ID NO:33 or its complement does not contain said complexes of two to seven marker DNAs.

Assignees

• Exact Sciences Dev Co Llc
• Mayo Found Medical Education & Res

Inventors

• Ahlquist David Alan
• Taylor William Russell
• Mahoney Douglas W
• Lidgard Graham P
• Allawi Hatim T
• Vaccaro Abram Michael

Classifications

• C12Q2600/166

  Oligonucleotides used as internal standards, controls or normalisation probes · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/154

  Methylation markers · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title