Methods and Kits for Methylation Detection

Abstract

Ligation-based methods and kits are disclosed for determining the degree of methylation of one or more target nucleotides. In certain embodiments, the methylation status of one or more target nucleotides is determined by generating misligation products. In certain embodiments, at least one target nucleotide is amplified prior to the ligation reaction. In certain embodiments, at least one ligation product, at least one ligation product surrogate, at least one misligation product, at least one misligation product surrogate, or combinations thereof are amplified. In certain embodiments, one or more ligation probes comprise at least one nucleotide analog, at least one Modification, at least one mismatched nucleotide, or combinations thereof.

1 - 69 . (canceled) 
     
     
         70 . A kit for determining the degree of methylation of at least one target nucleotide in at least one target nucleic acid sequence, comprising at least one ligation probe set and at least one ligation agent. 
     
     
         71 . The kit of  claim 79 , wherein the at least one thermostable ligase comprises Afu ligase. 
     
     
         72 . The kit of  claim 70 , further comprising: at least one amplifying agent; at least one primer; at least one reporter group; at least one reporter probe; at least one mobility modifier moiety; at least one hybridization tag; at least one hybridization tag complement; or combination thereof. 
     
     
         73 . The kit of  claim 72 , wherein the at least one amplifying agent comprises at least one thermostable polymerase. 
     
     
         74 . A kit for determining the degree of methylation of at least one target nucleotide comprising: at least one means for ligating; at least one means for amplifying; at least one means for separating; at least one means for digesting; at least one means for quantifying, or combination thereof. 
     
     
         75 - 76 . (canceled) 
     
     
         77 . The kit of  claim 70 , wherein the at least one ligation agent is a ligase. 
     
     
         78 . The kit of  claim 77 , wherein the at least one ligation agent is a DNA or an RNA ligase. 
     
     
         79 . The kit of  claim 77 , wherein the ligase is at least one thermostable ligase. 
     
     
         80 . The kit of  claim 79 , wherein the thermostable ligase is selected from group consisting of  Thermus species  ligase, Pfu ligase, and Afu ligase. 
     
     
         81 . The kit of  claim 73 , wherein the at least one thermostable polymerase is selected from the group consisting of Taq polymerase, Tfl polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, AMPLITAQ GOLD polymerase, 9° N m ™ polymerase, VENT R  DNA polymerase, DEEP VENT R  DNA polymerase, and UlTma polymerase. 
     
     
         82 . The kit of  claim 74 , wherein the thermostable polymerase is AMPLITAQ GOLD polymerase.