Analysis of methylation status and copy number

Abstract

A method of diagnosing a disease associated with a DNA repeat sequence is disclosed. The method comprises: (a) determining the number of repeats of a DNA sequence in DNA molecules of a sample of the subject; and (b) determining the CpG methylation status of the DNA molecules, wherein the number of repeats of the DNA sequence and the CpG methylation status of the DNA molecules is indicative of the disease.

What is claimed is: 
     
         1 . A method of diagnosing a disease associated with a DNA repeat sequence comprising:
 (a) determining the number of repeats of a DNA sequence in DNA molecules of a sample of the subject by attaching to the DNA molecules a detectable moiety which labels said repeats of said DNA sequence using a sequence-specific DNA labeling agent; and   (b) determining the CpG methylation status of said DNA molecules, wherein the number of repeats of said DNA sequence and the CpG methylation status of said DNA molecules is indicative of the disease.   
     
     
         2 . The method of  claim 1 , wherein step (b) is effected by contacting said DNA molecules with a methyltransferase (MTase) enzyme in the presence of a co-factor which is labeled with a detectable moiety under conditions which brings about transfer of said detectable moiety from said co-factor to said DNA molecules. 
     
     
         3 . The method of  claim 2 , further comprising extending the DNA molecules following said contacting. 
     
     
         4 . The method of  claim 3 , wherein said extending is linearly extending. 
     
     
         5 . The method of  claim 1 , wherein said sequence specific DNA labeling agent is a nicking enzyme. 
     
     
         6 . The method of  claim 5 , wherein said nicking enzyme is selected from the group consisting of Nb.BsmI, Nb.BvvCI, Nb.BsrDI, Nb.BssSI, Nb.Btsl, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI and Nt.BstNBI; or
 said nicking enzyme is selected from the group consisting of Cas9, TALE and ZFN nickase.   
     
     
         7 . The method of  claim 5 , wherein said nicking enzyme is selected from the group consisting of Cas9, TALE and ZFN nickase. 
     
     
         8 . The method of  claim 1 , wherein said sequence-specific DNA labeling agent is a CpG-methylation insensitive DNA MTase. 
     
     
         9 . The method of  claim 8 , wherein said CpG-methylation insensitive DNA methyltransferase has a double-stranded recognition sequence which does not contain the 5′-CG-3′ sequence. 
     
     
         10 . The method of  claim 9 , wherein said CpG-methylation insensitive DNA methyltransferase is a DNA adenine methylase (Dam), M.EcoDam or a derivative thereof or M.BseCI or a derivative thereof. 
     
     
         11 . A kit comprising:
 (i) a methyltransferase enzyme;   (ii) a co-factor of said methyltransferase enzyme which is labeled with a detectable moiety; and   (iii) a sequence specific DNA labeling agent.   
     
     
         12 . The kit of  claim 11 , wherein said sequence specific DNA labeling agent is a nicking enzyme. 
     
     
         13 . The kit of  claim 12 , wherein said nicking enzyme is selected from the group consisting of Nb.BsmI, Nb.BvvCI, Nb.BsrDI, Nb.BssSI, Nb.Btsl, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI and Nt.BstNBI. 
     
     
         14 . The kit of  claim 11 , further comprising at least one additional component selected from the group consisting of a DNA polymerase enzyme, fluorescent nucleotides and a DNA ligase enzyme. 
     
     
         15 . A method of analyzing the methylation status of a CpG site along a DNA molecule comprising:
 (a) contacting the DNA with a methyltransferase enzyme in the presence of a co-factor which is labeled with a detectable moiety under conditions which brings about transfer of said detectable moiety from said co-factor to said DNA;   (b) extending the DNA molecule; and   (c) detecting said detectable moiety, wherein the presence of the detectable moiety is indicative of a non-methylated CpG site.   
     
     
         16 . The method of  claim 15 , further comprising attaching to the DNA molecule an additional detectable moiety which labels a sequence of said DNA which is not a CpG site using a DNA labeling agent and wherein said DNA labeling agent is a sequence-specific DNA labeling agent. 
     
     
         17 . The method of  claim 16 , wherein said sequence-specific DNA labeling agent is a nicking enzyme. 
     
     
         18 . The method of  claim 16 , wherein said sequence-specific DNA labeling agent is a CpG-methylation insensitive DNA MTase. 
     
     
         19 . The method of  claim 18 , wherein said CpG-methylation insensitive DNA methyltransferase has a double-stranded recognition sequence which does not contain the 5′-CG-3′ sequence. 
     
     
         20 . The method of  claim 19 , wherein said CpG-methylation insensitive DNA methyltransferase is a DNA adenine methylase (Dam); or a M.EcoDam or a derivative thereof; or a M.BseCI or a derivative thereof. 
     
     
         21 . A mammalian DNA molecule comprising at least two different detectable moieties, wherein a first detectable moiety of said at least two different detectable moieties specifically labels unmethylated CpG sites and a second detectable moiety of said at least two different detectable moieties specifically labels a DNA sequence other than said CpG site, wherein the DNA molecule is a genomic DNA molecule.