Methylated control dna

Abstract

Provided herein is technology relating compositions and methods for analysis of methylated DNA from a subject. The technology also relates to use of endogenous methylated DNAs as internal controls for marker gene methylation assays.

1 . A composition, comprising:
 bisulfate-converted B3GALT6 DNA comprising SEQ ID NO:2 or the complement thereof and a pair of nucleic acid primers complementary to primer binding sites within the bisulfate-converted B3GALT6 DNA.   
     
     
         2 . The composition of  claim 1 , wherein said pair of nucleic acid primers are complementary to primer binding sites within SEQ ID NO:2 and the complement thereof. 
     
     
         3 . The composition of  claim 1 , further comprising a detection probe oligonucleotide, wherein the detection probe oligonucleotide comprises a region that is hybridized to a region of said B3GALT6 DNA amplifiable using the pair of nucleic acid primers. 
     
     
         4 . The composition of  claim 3 , wherein the detection probe oligonucleotide comprises a region that is complementary to a portion of SEQ ID NO:2 or the complement thereof. 
     
     
         5 . The composition of  claim 4 , wherein said detection probe oligonucleotide comprises a reporter molecule comprising one or more of a fluorophore and a flap sequence. 
     
     
         6 .- 7 . (canceled) 
     
     
         8 . The composition of  claim 1 , further comprising one or more of:
 a FRET cassette   a FEN-1 endonuclease; and   a thermostable DNA polymerase.   
     
     
         9 .- 12 . (canceled) 
     
     
         13 . A kit, comprising:
 a) a pair of nucleic acid primers complementary to primer binding sites within bisulfite-converted B3GALT6 DNA; and   b) bisulfite reagent.   
     
     
         14 . The kit of  claim 13 , wherein the pair of nucleic acid primers are complementary to primer binding sites within SEQ ID NO:2 or a complement thereof. 
     
     
         15 . The kit of  claim 13 , further comprising at least one oligonucleotide selected from a capture oligonucleotide, a nucleic acid probe, and an invasive oligonucleotide. 
     
     
         16 .- 20 . (canceled) 
     
     
         21 . A method of characterizing a sample from a subject, comprising:
 a) treating DNA from a sample from the subject with a methylation-specific reagent to produce a treated DNA sample comprising converted methylation marker DNA and converted B3GALT6 DNA;   b) measuring an amount of at least one converted methylation marker DNA in the treated DNA sample;   c) measuring an amount of converted B3GALT6 DNA in the treated DNA sample;   
       and
 d) determining an amount of at least one methylation marker DNA in said sample from the subject by calculating the amount of said at least one converted methylation marker DNA as a percentage of the amount of said converted B3GALT6 DNA in the treated DNA sample. 
 
     
     
         22 . The method of  claim 21 , wherein measuring said amount of at least one converted methylation marker DNA and said amount of converted B3GALT6 DNA in said treated DNA sample comprises amplifying regions of said converted DNA using a pair of nucleic acid primers complementary to the converted methylation marker DNA and a pair of nucleic acid primers complementary to the converted B3GALT6 DNA, wherein said amplifying produces at least one amplified product comprising a region of said at least one methylation marker DNA, and an amplified product comprising a region of said B3GALT6 DNA. 
     
     
         23 . The method of  claim 21 , wherein said at least one converted methylation marker DNA comprises at least two converted methylation marker DNAs. 
     
     
         24 . The method of  claim 22 , wherein said amplified product comprising a region of said B3GALT6 DNA has a nucleic acid sequence comprising a region of SEQ ID NO:2, or a complement thereof. 
     
     
         25 . The method of  claim 21 , wherein said sample is a blood sample from a subject, wherein the amount of the converted methylation marker DNA in the blood sample is indicative of the fraction of the methylation marker DNA in the blood sample that is methylated, and wherein methylation of the methylation marker DNA in a solid tissue in the subject is indicative of a tumor in said tissue. 
     
     
         26 .- 27 . (canceled) 
     
     
         28 . The method of  claim 21 , wherein the wherein the methylation-specific reagent modifies methylated cytosine nucleotides to produce different nucleotides. 
     
     
         29 . The method of  claim 21 , wherein the methylation-specific reagent modifies unmethylated cytosine nucleotides to produce different nucleotides. 
     
     
         30 . The method of  claim 21 , wherein the methylation-specific reagent deaminates unmethylated cytosine nucleotides to produce deoxy uracil nucleotides. 
     
     
         31 . The method of  claim 30 , wherein the pair of nucleic acid primers complementary to converted B3GALT6 DNA are complementary to primer binding sites within SEQ ID NO:2 and the complement thereof. 
     
     
         32 . The method of  claim 30 , wherein the methylation-specific reagent comprises a bisulfite reagent.