Patent ID: 8993242
Filing Date: 2015-03-31
Classification: C12Q

Abstract:
1. A method for determining the effect of C-5 methylating one or more predetermined cytosine nucleotide residues of a deoxyribonucleic acid (DNA) on expression of a gene of interest comprising the steps of: a) denaturing a circular double-stranded DNA construct, wherein a cytosine residue of one of the strands of the circular DNA construct corresponds to the predetermined cytosine nucleotide residue and wherein the circular DNA construct comprises a nucleotide sequence identical to the gene of interest; b) hybridizing each of a pair of primers to separate strands of the denatured circular DNA construct, wherein each primer comprises a 5′ phosphate, and wherein at least one of the primers is C-5 methylated at a cytosine nucleotide residue thereof which is (i) immediately 5′ to a guanine nucleotide residue of the primer, and (ii) complementary to a guanine nucleotide residue of the strand of the circular DNA construct immediately 3′ to the cytosine nucleotide residue of the DNA construct which corresponds to the predetermined cytosine nucleotide residue; c) contacting the hybridized primers resulting from step b) with a DNA polymerase, deoxynucleoside triphosphates, and a plurality of copies of the pair of primers under conditions permitting a polymerase chain reaction to occur, thereby producing a plurality of nicked copies of the circular DNA construct, wherein each strand of each nicked copy incorporates a primer comprising a 5′ phosphate and wherein at least one strand of each nicked copy incorporates the C-5 methylated primer; d) contacting at least one copy of the plurality of nicked copies with a DNA ligase so as to form a phosphodiester bond between the 5′ phosphate of each incorporated primer of each strand and each respective 3′ end of each strand of the at least one copy so as to thereby form a copy of the circular DNA construct; e) contacting the copy of the circular DNA construct with a methyltransferase enzyme so as to thereby C-5 methylate the cytosine nucleotide residue immediately 5′ to the guanine nucleotide residue which is hybridized to the C-5 methylated cytosine nucleotide residue of the C-5 methylated primer incorporated into the copy of the circular DNA construct; f) transfecting a cell with the C-5 methylated circular DNA construct resulting from step e); and g) quantifying expression by the cell of the gene of interest, thereby determining the effect of C-5 methylating the one or more predetermined cytosine nucleotide residues of the deoxyribonucleic acid on expression of the gene of interest.