Dna methylation detection methods

Abstract

The present teachings provide DNA methylation quantification methods that avoid bisulfite treatment of DNA. Methylation-specific binding proteins (MeDNA binding proteins) and non-methylation specific binding proteins (non-MeDNA binding proteins) are employed in various embodiments to modulate the accessibility of nucleic acids to primer extension reactions. After selectively removing the target nucleic acids, the extension products can be analyzed and methylation quantitated. In some embodiments, the analysis comprises real-time PCR.

1 . A method of quantitating methylation in a target nucleic acid comprising;
 treating a target nucleic acid with a MeDNA binding protein, wherein the MeDNA binding protein forms a blocking complex with a methylated cytosine in the target nucleic acid, wherein the methylated cytosine in the target nucleic acid is near a first target specific primer binding site;   extending a first target specific primer hybridized to the first target specific primer binding site to form a target nucleic acid extension product;   degrading the target nucleic acid;   amplifying the target nucleic acid extension product;   determining the difference between the amount of the target nucleic acid with the amount of a control nucleic acid lacking a methylated cytosine; and,   quantitating methylation in the target nucleic acid.   
   
   
       2 . The method according to  claim 1  wherein the amplifying comprises a polymerase chain reaction comprising a first target specific primer and a second target specific primer. 
   
   
       3 . The method according to  claim 1  wherein the degrading comprises treating the target nucleic acid with a nuclease, wherein the target nucleic acid extension product is resistant to the nuclease due to a blocking moiety in the first target specific primer. 
   
   
       4 . A method of quantitating methylation in a target nucleic acid comprising;
 treating, in any order,
 (a) the target nucleic acid with a MeDNA binding protein, wherein the MeDNA binding protein forms a blocking complex with a methylated cytosine in the target nucleic acid, wherein the methylated cytosine is disposed between a first target specific primer binding site and a second target specific primer binding site; and, 
 (b) a control nucleic acid with a MeDNA binding protein, wherein the MeDNA binding protein fails to form a blocking complex with an unmethylated cytosine in the control nucleic acid, wherein the non-methylated cytosine is disposed between a first control specific primer binding site and a second control specific primer binding site; 
   extending, in any order,
 (a) a first target specific primer hybridized to the first target specific primer binding site to form a target nucleic acid extension product; and, 
 (b) a first control specific primer hybridized to the first control specific primer binding site to form a control nucleic acid extension product; 
   degrading, in any order,
 (a) the target nucleic acid; and, 
 (b) the control nucleic acid; 
   amplifying, in any order,   (a) the target nucleic acid extension product in a polymerase chain reaction comprising a first target specific primer and a second target specific primer; and,   (b) the control nucleic acid extension product in a polymerase chain reaction comprising a first control specific primer and a second control specific primer;   determining the difference between the amount of target nucleic acid with the amount of control nucleic acid; and,   quantitating methylation in the target nucleic acid.   
   
   
       5 . The method of  claim 4  wherein the determining comprises;
 measuring, in any order,
 (a) a first Ct value associated with the amount of the target nucleic acid, and, 
 (b) a second Ct value associated with the amount of the control nucleic acid; and, 
   quantitating methylation in the target nucleic by comparing the first Ct value with the second Ct value.   
   
   
       6 . The method of  claim 4  wherein the first Ct value is higher than the second Ct value, and the target nucleic acid is more methylated than the control nucleic acid. 
   
   
       7 . The method of  claim 4  wherein the first Ct value is lower than the second Ct value, and the target nucleic acid is less methylated than the control nucleic acid. 
   
   
       8 . The method of  claim 4  wherein the target nucleic acid and the control nucleic acid comprise the same first primer binding site and the same second primer binding site. 
   
   
       9 . The method according to  claim 4  wherein the target nucleic acid is amplified in a separate reaction vessel from the control nucleic acid. 
   
   
       10 . The method of  claim 9  wherein the target nucleic acid and the control nucleic acid comprise the same first primer binding site and the same second primer binding site and are amplified with a common first primer and a common second primer. 
   
   
       11 . The method according to  claim 4  wherein the target nucleic acid is amplified in a same reaction vessel as the control nucleic acid. 
   
   
       12 . The method of  claim 11  wherein the target nucleic acid and the control nucleic acid comprise a different first primer binding site and a different second primer binding site and are amplified with a different first primer and a different second primer. 
   
   
       13 . The method according to  claim 4  wherein the quantitating comprises measuring an interchelating dye. 
   
   
       14 . The method according to  claim 4  wherein the determining comprises;
 measuring displacement of a target sequence specific probe, wherein the target sequence specific probe hybridizes to a region of the target nucleic acid extension product, or complement to the target nucleic acid extension product, disposed between the first target specific primer binding site and the second target specific primer binding site;   measuring displacement of a control sequence specific probe, wherein the control sequence specific probe hybridizes to a region of the control nucleic acid extension product, or complement to the control nucleic acid extension product, disposed between the first control specific primer binding site and the second control specific primer binding site.   
   
   
       15 . The method according to  claim 4 , wherein the treating with the MeDNA binding protein further comprises a cofactor. 
   
   
       16 . The method according to  claim 15  wherein the cofactor is selected from the group consisting of S-adenosylmethionine, S-adenosylhomocysteine and sinefungin. In a preferred embodiment, S-adenosylhomocycteine is used. 
   
   
       17 . The method according to  claim 4  wherein the first primer hybridizes upstream from the blocking complex formed by the MeDNA binding protein and the methylated cytosine in the target nucleic acid, and further extension of the first primer is blocked. 
   
   
       18 . The method according to  claim 4  wherein the first primer hybridizes on the methylated cytosine on the blocking complex formed by the MeDNA binding protein and the methylated cytosine in the target nucleic acid, and initial extension of the first primer is blocked. 
   
   
       19 . The method according to  claim 4  wherein the first primer does not hybridize to the methylated cytosine on the blocking complex formed by the MeDNA binding protein and the methylated cytosine in the target nucleic acid. 
   
   
       20 . A kit for quantitating methylation in a target nucleic acid comprising;
 a Me-DNA binding protein;   a first target specific primer;   a second target specific primer;   a first control specific primer;   a second control specific primer; and,   a polymerase.   
   
   
       21 . The kit according to  claim 20  wherein;
 the first control specific primer is a different sequence from the first target specific primer; and,   the second control specific primer is a different sequence from the second target specific primer.   
   
   
       22 . The kit according to  claim 20  wherein;
 the first control specific primer is a same sequence as the first target specific primer; and,   the second control specific primer is a same sequence as the second target specific primer.   
   
   
       23 . The kit according to  claim 20  further comprising a control sequence specific probe. 
   
   
       24 . The kit according to  claim 23  further comprising a target sequence specific probe, wherein the control sequence specific probe is a different sequence from the target sequence specific probe. 
   
   
       25 . The kit according to  claim 23  further comprising a target sequence specific probe, wherein the control sequence specific probe is a same sequence as the target sequence specific probe. 
   
   
       26 . The kit according to  claim 20  further comprising a cofactor. 
   
   
       27 . The kit according to  claim 26  wherein the cofactor is selected from the group consisting of S-adenosylmethionine, S-adenosylhomocysteine and sinefungin. 
   
   
       28 . A method of quantitating methylation in a target nucleic acid comprising;
 treating a control nucleic acid with a non-MeDNA binding protein, wherein the non-MeDNA binding protein forms a blocking complex with an unmethylated cytosine in the control nucleic acid, wherein the unmethylated cytosine in the control nucleic acid is near a first control specific primer binding site;   extending a first primer hybridized to the first primer binding site to form a control nucleic acid extension product;   degrading the control nucleic acid;   amplifying the control nucleic acid extension product in a polymerase chain reaction comprising a first control specific primer and a second control specific primer;   determining the difference between the amount of the control nucleic acid with the amount of a target nucleic acid containing a methylated cytosine; and,   quantitating methylation in the target nucleic acid.   
   
   
       29 . The method according to  claim 28  wherein the amplifying comprises a polymerase chain reaction comprising a first target specific primer and a second target specific primer. 
   
   
       30 . The method according to  claim 28  wherein the degrading comprises treating the target nucleic acid with a nuclease, wherein the target nucleic acid extension product is resistant to the nuclease due to a blocking moiety in the first target specific primer. 
   
   
       31 . A method of quantitating methylation in a target nucleic acid comprising;
 treating, in any order,
 (a) the control nucleic acid with a non-MeDNA binding protein, wherein the non-MeDNA binding protein forms a blocking complex with an unmethylated cytosine in the control nucleic acid lacking, wherein the unmethylated cytosine is disposed between a first control-specific primer binding site and a second control-specific primer binding site; and, 
 (b) a target nucleic acid with a non-MeDNA binding protein, wherein the non-MeDNA binding protein fails to form a blocking complex with a a methylated cytosine in the target nucleic acid, wherein the methylated cytosine is disposed between a first target specific primer binding site and a second target specific primer binding site; 
   extending, in any order,
 (a) a first target specific primer hybridized to the first target specific primer binding site to form a target nucleic acid extension product; and, 
 (b) a first control specific primer hybridized to the first control specific primer binding site to form a control nucleic acid extension product; 
   degrading, in any order,
 (a) the target nucleic acid; and, 
 (b) the control nucleic acid; 
   amplifying, in any order,
 (a) the target nucleic acid extension product in a polymerase chain reaction comprising a first target specific primer and a second target specific primer; and, 
 (b) the control nucleic acid extension product in a polymerase chain reaction comprising a first control specific primer and a second control specific primer; 
   determining the difference between the amount of target nucleic acid with the amount of control nucleic acid; and,   quantitating methylation in the target nucleic acid.   
   
   
       32 . The method of  claim 31  wherein the determining comprises;
 measuring, in any order,
 (a) a first Ct value associated with the amount of the target nucleic acid, and, 
 (b) a second Ct value associated with the amount of the control nucleic acid; and, 
   quantitating methylation in the target nucleic by comparing the first Ct value with the second Ct value.   
   
   
       33 . The method of  claim 31  wherein the first Ct value is higher than the second Ct value, and the target nucleic acid is less methylated than the control nucleic acid. 
   
   
       34 . The method of  claim 31  wherein the first Ct value is lower than the second Ct value, and the target nucleic acid is more methylated than the control nucleic acid. 
   
   
       35 . The method of  claim 31  wherein the target nucleic acid and the control nucleic acid comprise the same first primer binding site and the same second primer binding site. 
   
   
       36 . The method according to  claim 31  wherein the target nucleic acid is amplified in a separate reaction vessel from the control nucleic acid. 
   
   
       37 . The method of  claim 36  wherein the target nucleic acid and the control nucleic acid comprise the same first primer binding site and the same second primer binding site and are amplified with a common first primer and a common second primer. 
   
   
       38 . The method according to  claim 31  wherein the target nucleic acid is amplified in a same reaction vessel as the control nucleic acid. 
   
   
       39 . The method of  claim 38  wherein the target nucleic acid and the control nucleic acid comprise a different first primer binding site and a different second primer binding site and are amplified with a different first primer and a different second primer. 
   
   
       40 . The method according to  claim 31  wherein the quantitating comprises measuring an interchelating dye. 
   
   
       41 . The method according to  claim 31  wherein the determining comprises;
 measuring displacement of a target sequence specific probe, wherein the target sequence specific probe hybridizes to a region of the target nucleic acid extension product, or complement to the target nucleic acid extension product, disposed between the first target specific primer binding site and the second target specific primer binding site;   measuring displacement of a control sequence specific probe, wherein the control sequence specific probe hybridizes to a region of the control nucleic acid extension product, or complement to the control nucleic acid extension product, disposed between the first control specific primer binding site and the second control specific primer binding site.   
   
   
       42 . The method according to  claim 31 , wherein the treating with the non-MeDNA binding protein further comprises a cofactor. 
   
   
       43 . The method according to  claim 42  wherein the cofactor is SELECTED FROM THE GROUP CONSISTING OF S-adenosylmethionine, S-adenosylhomocysteine and sinefungin. In a preferred embodiment, S-adenosylhomocycteine is used. 
   
   
       44 . The method according to  claim 31  wherein the first primer hybridizes upstream from the blocking complex formed by the non-MeDNA binding protein and the unmethylated cytosine in the control nucleic acid, and further extension of the first primer is blocked. 
   
   
       45 . The method according to  claim 31  wherein the first primer hybridizes on the unmethylated cytosine on the blocking complex formed by the non-MeDNA binding protein and the unmethylated cytosine in the control nucleic acid, and initial extension of the first primer is blocked. 
   
   
       46 . The method according to  claim 31  wherein the first primer does not hybridize to the unmethylated cytosine on the blocking complex formed by the non-MeDNA binding protein and the unmethylated cytosine in the target nucleic acid. 
   
   
       47 . A kit for quantitating methylation in a target nucleic acid comprising;
 a non-MeDNA binding protein;   a first target specific primer;   a second target specific primer;   a first control specific primer;   a second control specific primer; and,   a polymerase.   
   
   
       48 . The kit according to  claim 47  wherein;
 the first control specific primer is a different sequence from the first target specific primer; and,   the second control specific primer is a different sequence from the second target specific primer.   
   
   
       49 . The kit according to  claim 47  wherein;
 the first control specific primer is a same sequence as the first target specific primer; and,   the second control specific primer is a same sequence as the second target specific primer.   
   
   
       50 . The kit according to  claim 47  further comprising a control sequence specific probe. 
   
   
       51 . The kit according to  claim 50  further comprising a target sequence specific probe, wherein the control sequence specific probe is a different sequence from the target sequence specific probe. 
   
   
       52 . The kit according to  claim 50  further comprising a target sequence specific probe, wherein the control sequence specific probe is a same sequence as the target sequence specific probe. 
   
   
       53 . The kit according to  claim 47  further comprising a cofactor. 
   
   
       54 . The kit according to  claim 53  wherein the cofactor is selected from the group consisting of S-adenosylmethionine, S-adenosylhomocysteine and sinefungin.