# US 11,208,680 B2 · Nucleic acid control molecules from non-human organisms > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,208,680 B2 - Document ID: US11208680B2 - Status: Granted - Kind: B2 - Application number: 16/318,578 - Filed: 2017-07-19 - Published: 2020-02-06 - Granted: 2021-12-28 - Earliest claimed: 2016-07-19 - Assignees: Exact Sciences Development Company, LLC - Applicants: Exact Sciences Development Company, LLC - Inventors: Hatim T. Allawi; Graham P. Lidgard; Brian Aizenstein; Melissa M. Gray - Web page: https://patentgrep.com/patent/US11208680B2 ## CPC - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) - C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers} - C12Q 2600/154: Methylation markers - C12Q 2600/16: Primer sets for multiplex assays - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention provides synthetic DNA strands that find use as process controls in DNA processing and nucleic acid testing methods. In particular, provided herein are synthetic methylated DNA strands of known composition for use as control molecules in DNA testing, e.g., of mutations and/or methylation of DNA isolated from non-fish samples, such as human samples. ## Claims ### Claim 1 A composition comprising a) isolated methylated zebrafish DNA or synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence, and b) a second component, in a mixture, wherein said second component is selected from: methylated DNA from a biological sample from a human; a eukaryotic cell from a human; and a biological sample from a human. ### Claim 2 (depends on 1) The composition of claim 1, further comprising at least one component from the group consisting of: a bacterial, phage, viral, archaeal, or non-fish eukaryotic nucleic acid polymerase; and a bacterial, phage, archaeal, or non-fish eukaryotic DNA modifying enzyme. ### Claim 3 (depends on 2) The composition of claim 2, wherein said nucleic acid polymerase is a DNA polymerase. ### Claim 4 (depends on 3) The composition of claim 3, wherein said DNA polymerase is a thermostable DNA polymerase. ### Claim 5 (depends on 2) The composition of claim 2, wherein said DNA modifying enzyme comprises a ligase, an exonuclease, and/or an endonuclease. ### Claim 6 (depends on 5) The composition of claim 5, wherein said endonuclease is a flap endonuclease. ### Claim 7 (depends on 1) The composition of claim 1, further comprising at least one feature from the group of features consisting of: i) said isolated methylated zebrafish DNA or said synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence comprises at least a portion of the zebrafish rassf1 gene; ii) said isolated methylated zebrafish DNA or said synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence comprises at least a portion of SEQ ID NO:1 or its complement; iii) said isolated methylated zebrafish DNA or said synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence comprises at least a portion of SEQ ID NO:2 or a complement thereof, or SEQ ID NO:3 or a complement thereof; and iv) said synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence comprises at least one oligonucleotide from the group consisting of: an oligonucleotide having the sequence of SEQ ID NO: 11; and an oligonucleotide having the sequence of SEQ ID NO:12. ### Claim 8 (depends on 1) The composition of claim 1, wherein said isolated methylated zebrafish DNA or synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence is bisulfite-treated DNA. ### Claim 9 (depends on 1) The composition of claim 1, wherein said biological sample from a human comprises one or more of blood, serum, plasma, tissue, stool, or sputum. ### Claim 10 A method of treating a biological sample containing methylated DNA from a human subject, comprising a) combining isolated methylated zebrafish DNA or synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence with said biological sample in a mixture; and b) treating said mixture to purify DNA from said mixture. ### Claim 11 (depends on 10) The method of claim 10, wherein said treating comprises binding DNA to a support. ### Claim 12 (depends on 11) The method of claim 11, wherein said support comprises a silica particle. ### Claim 13 (depends on 10) The method of claim 10, further comprising treating DNA purified from said mixture with a bisulfite reagent. ### Claim 14 (depends on 10) The method of claim 10, further comprising detecting DNA purified from said mixture with a nucleic acid detection assay. ### Claim 15 (depends on 14) The method of claim 14, wherein detecting with said nucleic acid detection assay comprises detecting DNA from said biological sample and said methylated zebrafish DNA or said synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence. ### Claim 16 (depends on 10) The method of claim 10, further comprising at least one feature from the group of features consisting of: i) said isolated methylated zebrafish DNA or said synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence comprises at least a portion of the zebrafish rassf1 gene; ii) said isolated methylated zebrafish DNA or said synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence comprises at least a portion of SEQ ID NO:1 or its complement; iii) said isolated methylated zebrafish DNA or said synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence comprises at least a portion of SEQ ID NO:2 or a complement thereof, or SEQ ID NO:3 or a complement thereof; and iv) said synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence comprises at least one oligonucleotide from the group consisting of: an oligonucleotide having the sequence of SEQ ID NO: 11; and an oligonucleotide having the sequence of SEQ ID NO:12.