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 kit for analyzing methylated DNA from a biological sample from a human, the kit comprising:
a) synthetic methylated DNA comprising a zebrafish DNA nucleotide sequence;
b) an oligonucleotide comprising a region complementary to said synthetic methylated DNA;
c) an oligonucleotide comprising a region complementary to methylated DNA from a human; and
d) a methylation-specific reagent system.
2. The kit of claim 1 , further comprising one or more components selected from the group consisting of:
i) a silica particle or silica-coated particle;
ii) a chaotropic agent;
iii) a buffer;
iv) a nucleic acid polymerase;
v) a DNA-modifying enzyme; and
vi) a FRET cassette.
3. The kit of claim 2 , wherein the nucleic acid polymerase comprises a bacterial, phage, viral, archaeal, or non-fish eukaryotic nucleic acid polymerase.
4. The kit of claim 3 , wherein said nucleic acid polymerase is a DNA polymerase.
5. The kit of claim 4 , wherein said DNA polymerase is a thermostable DNA polymerase.
6. The kit of claim 2 , further comprising a bacterial, phage, archaeal, or non-fish eukaryotic DNA modifying enzyme.
7. The kit of claim 6 , wherein said DNA modifying enzyme comprises a ligase, an exonuclease, and/or an endonuclease.
8. The kit of claim 7 , wherein said endonuclease is a flap endonuclease.
9. The kit of claim 8 , wherein the flap endonuclease comprises a FEN-1 endonuclease.
10. The kit of claim 9 , wherein the FEN-1 endonuclease is an archaeal FEN-1 endonuclease.
11. The kit of claim 1 , wherein the methylation-specific reagent system modifies unmethylated cytosine nucleotides to produce different nucleotides.
12. The kit of claim 11 , wherein the methylation-specific reagent system deaminates unmethylated cytosine nucleotides to produce deoxy uracil nucleotides.
13. The kit of claim 12 , wherein the methylation-specific reagent system comprises a bisulfate reagent.
14. The kit of claim 1 , wherein the methylation-specific reagent system modifies methylated cytosine nucleotides to produce different nucleotides.
15. The kit of claim 1 , wherein the synthetic methylated DNA comprises at least a portion of SEQ ID NO:1 or its complement or SEQ ID NO:2 or its complement.
16. The kit of claim 15 , wherein the synthetic methylated DNA comprises an oligonucleotide having the sequence of SEQ ID NO:11 and/or an oligonucleotide having the sequence of SEQ ID NO:12.
17. The kit of claim 16 , wherein the synthetic methylated DNA consists essentially of an oligonucleotide having the sequence of SEQ ID NO:11.
18. The kit of claim 1 , comprising an oligonucleotide comprising a region complementary to the synthetic methylated DNA after modification of the synthetic methylated DNA with the methylation-specific reagent system.
19. The kit of claim 1 , comprising an oligonucleotide comprising a region complementary to the methylated DNA from a human after modification of the methylated DNA from a human with the methylation-specific reagent system.