Nucleic acid control molecules from non-human organisms
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.
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.
2. 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.
3. The composition of claim 2 , wherein said nucleic acid polymerase is a DNA polymerase.
4. The composition of claim 3 , wherein said DNA polymerase is a thermostable DNA polymerase.
5. The composition of claim 2 , wherein said DNA modifying enzyme comprises a ligase, an exonuclease, and/or an endonuclease.
6. The composition of claim 5 , wherein said endonuclease is a flap endonuclease.
7. 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.
8. 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.
9. The composition of claim 1 , wherein said biological sample from a human comprises one or more of blood, serum, plasma, tissue, stool, or sputum.
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.
11. The method of claim 10 , wherein said treating comprises binding DNA to a support.
12. The method of claim 11 , wherein said support comprises a silica particle.
13. The method of claim 10 , further comprising treating DNA purified from said mixture with a bisulfite reagent.
14. The method of claim 10 , further comprising detecting DNA purified from said mixture with a nucleic acid detection assay.
15. 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.
16. 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.