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 - 39 . (canceled)
40 . A composition comprising a mixture of DNA treated with a methylation-specific reagent system to form a treated DNA mixture, wherein the treated DNA mixture comprises:
a) isolated methylated zebrafish DNA or synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence, and
b) one or more of:
i) methylated DNA from a biological sample from a human, and
ii) DNA from a eukaryotic cell from a human;
wherein the methylation-specific reagent system modifies nucleotides in DNA based on their methylation state to produce different nucleotides.
41 . The composition of claim 40 , wherein the methylation-specific reagent system modifies unmethylated cytosine nucleotides to produce different nucleotides.
42 . The composition of claim 40 , wherein the methylation-specific reagent system deaminates unmethylated cytosine nucleotides to produce deoxy uracil nucleotides.
43 . The composition of claim 42 , wherein the methylation-specific reagent system comprises a bisulfite reagent.
44 . The composition of claim 40 , wherein the methylation-specific reagent system modifies methylated cytosine nucleotides to produce different nucleotides.
45 . The composition of claim 40 , further comprising:
c) an oligonucleotide comprising a region complementary to treated methylated zebrafish DNA or treated synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence; and
d) an oligonucleotide comprising a region complementary to treated methylated DNA from a human.
46 . The composition of claim 40 , wherein the isolated methylated zebrafish DNA or the synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence comprises at least a portion of zebrafish rassf1 gene DNA.
47 . The composition of claim 40 , wherein the treated DNA mixture further comprises at least one component from the group consisting of:
i) a bacterial, phage, viral, archaeal, or non-fish eukaryotic nucleic acid polymerase; and
ii) a bacterial, phage, archaeal, or non-fish eukaryotic DNA modifying enzyme.
48 . The composition of claim 47 , wherein the nucleic acid polymerase is a thermostable DNA polymerase.
49 . The composition of claim 47 , wherein said DNA modifying enzyme comprises a ligase, an exonuclease, an endonuclease, and a FEN-1 flap endonuclease.
50 . The composition of claim 40 , wherein said biological sample from a human comprises one or more of blood, serum, plasma, tissue, stool, and sputum.
51 . A method of treating methylated DNA from a human subject, comprising:
a) combining isolated methylated zebrafish DNA or synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence with a sample comprising methylated DNA from a human in a mixture; and
b) treating the mixture with a methylation-specific reagent system to form a treated DNA mixture,
wherein the methylation-specific reagent system modifies nucleotides in DNA based on their methylation state to produce different nucleotides.
52 . The method of claim 51 , wherein the methylation-specific reagent system modifies unmethylated cytosine nucleotides to produce different nucleotides.
53 . The method of claim 51 , wherein the methylation-specific reagent system deaminates unmethylated cytosine nucleotides to produce deoxy uracil nucleotides.
54 . The method of claim 51 , wherein the methylation-specific reagent system comprises a bisulfite reagent.
55 . The method of claim 51 , wherein the sample comprising methylated DNA from a human comprises a biological sample containing methylated DNA from a human subject, and wherein prior to step b), the method comprises:
i) combining isolated methylated zebrafish DNA or synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence with the biological sample in a combined sample; and
ii) treating said combined sample to purify a DNA from the combined sample to provide the mixture of step a).
56 . The method of claim 51 , wherein the sample comprising methylated DNA from a human comprises one or more of:
i) DNA purified from a biological sample from a human; and
ii) DNA purified from a eukaryotic cell from a human.
57 . The method of claim 51 , wherein the isolated methylated zebrafish DNA or the synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence comprises at least a portion of zebrafish rassf1 gene DNA.
58 . The method of claim 51 , further comprising:
c) detecting treated isolated methylated zebrafish DNA or synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence and treated methylated DNA from a human in the treated DNA mixture.
59 . A kit for analyzing methylated DNA from a biological sample from a human or DNA from a eukaryotic cell from a human, the kit comprising:
a) isolated methylated zebrafish DNA or synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence;
b) a methylation-specific reagent system that modifies nucleotides in DNA based on their methylation state to produce different nucleotides;
c) an oligonucleotide comprising a region complementary to the DNA of a) after treatment with the methylation-specific reagent system;
d) an oligonucleotide comprising a region complementary to a methylated DNA from a human after treatment of the methylated DNA from a human with the methylation-specific reagent system; and
e) one or more bacterial, phage, viral, archaeal, or non-fish eukaryotic enzymes selected from:
i) a nucleic acid polymerase;
ii) a ligase,
iii) an exonuclease;
iv) an endonuclease; and
v) an archaeal FEN-1 endonuclease.