Synthetic nucleic acid control molecules

Abstract

The present invention provides synthetic DNA strands that find use as controls or in nucleic acid testing methods. In particular, provided herein are synthetic DNA strands of known composition for use as control molecules in stool DNA testing, e.g., of mutations and/or methylation of DNA isolated from stool samples.

1 .- 78 . (canceled) 
     
     
         79 . A method of characterizing a methylation assay, comprising:
 i) providing a run control composition that comprises:
 a) a number of copies of a first synthetic DNA fragment comprising a sequence of a portion of a human biomarker DNA, wherein the sequence is synthesized to comprise a sequence region containing a number and pattern of cytosines and cytosine-phosphate-guanosine (CpG) dinucleotides in which each of the cytosines in the CpG dinucleotides in the pattern comprises a 5-methyl; 
 b) a number of copies of a second synthetic DNA fragment comprising the same sequence of the same portion of the human biomarker DNA as the first synthetic DNA, and comprising a sequence region comprising the same number and pattern of cytosines and CpG dinucleotides as the sequence region in the first synthetic DNA fragment, wherein none of the cytosines in the pattern comprises a 5-methyl; and 
 c) fish DNA; and 
   ii) applying the methylation assay to the run control composition, wherein cytosines in the CpG dinucleotides in the pattern are detected.   
     
     
         80 . The method of  claim 79 , wherein cytosines in the CpG dinucleotides in the pattern that do not comprise a 5-methyl are detected. 
     
     
         81 . The method of  claim 79 , wherein cytosines in the CpG dinucleotides in the pattern that comprise a 5-methyl are detected. 
     
     
         82 . The method of  claim 79 , wherein the methylation assay comprises treating the run control composition with a reagent that converts unmethylated cytosine bases to uracil bases. 
     
     
         83 . The method of method of  claim 82 , wherein the reagent comprises bisulfite. 
     
     
         84 . The method of  claim 82 , wherein the methylation assay comprises assaying treated run control DNA to detect a number of copies of treated first synthetic DNA fragment and a number of copies of treated second synthetic DNA fragment. 
     
     
         85 . The method of  claim 84 , wherein the number of copies of treated first synthetic DNA fragment and the number of copies of treated second synthetic DNA fragment are detected in separate reaction mixtures. 
     
     
         86 . The method of  claim 85 , wherein the reaction mixtures comprise one or more of DNA polymerase, deoxynucleoside triphosphates, amplification buffer, primer oligonucleotides, probe oligonucleotide, and magnesium salt. 
     
     
         87 . The method of  claim 79 , wherein the methylation assay comprises an invasive cleavage assay. 
     
     
         88 . The method of  claim 87 , wherein the invasive cleavage assay is a flap assay. 
     
     
         89 . The method of  claim 88 , wherein the flap assay comprises using one or more reagents selected from the group consisting of an invasive oligonucleotide, a flap oligonucleotide, a flap endonuclease, and a FRET cassette. 
     
     
         90 . The method of  claim 79 , wherein the first synthetic DNA fragment and the second synthetic DNA fragment are double-stranded. 
     
     
         91 . The method of  claim 79 , wherein the first synthetic DNA fragment and the second synthetic DNA fragment each further comprise a second sequence region containing a CpG dinucleotide in which the cytosine does not comprise a 5-methyl. 
     
     
         92 . The method of  claim 79 , wherein the run control composition comprises at least 50 μg/mL fish DNA. 
     
     
         93 . The method of  claim 79 , wherein the run control composition further comprises a DNA stabilization buffer comprising Tris-HCl, EDTA, and NaCl. 
     
     
         94 . The method of  claim 93 , wherein the run control composition has a volume and wherein the DNA stabilization buffer is 80% of the volume of the run control composition, and wherein the DNA stabilization buffer comprises 500 mM Tris-HCl, 150 mM EDTA, and 10 mM NaCl, pH 9. 
     
     
         95 . The method of  claim 79 , wherein the run control composition further comprises a synthetic DNA fragment comprising a sequence from a human reference gene.