| 主权项 |
1. A method of removing amplicons of a non target nucleic acid having one or more cytosines that are methylated from a sample wherein the sample comprises amplicons of the non target nucleic acid and genomic DNA that includes a target nucleic acid to be serially amplified, the target nucleic acid having one or more methylated cytosines, comprising
a) contacting the sample with a composition comprising (i) deoxynucleotide triphophates (dNTPs) comprising dATP, dTTP, dGTP, and dCTP wherein one or more of the dCTP nucleotides are methylated with a first methyl group (ii) a nucleic acid polymerase, (iii) one or more primers that are complementary to a portion of the target nucleic acid, and (iv) a first methyl specific restriction enzyme that is capable of being deactivated and that digests nucleic acids comprising nucleotides that are methylated with the first methyl group, thereby producing a combination; b) maintaining the combination under conditions in which the amplicons of the non target nucleic acid are digested by the first methyl specific restriction enzyme prior to amplification of the genomic DNA that includes the target nucleic acid; c) amplifying the genomic DNA that includes the target nucleic acid, thereby producing amplicons of the target nucleic acid having one or more nucleotides that are methylated with the first methyl group; d) contacting the amplicons of the target nucleic acid with a composition comprising (i) deoxynucleotide triphophates (dNTPs) comprising dATP, dTTP, dGTP, and dCTP wherein one or more of the dCTP nucleotides are methylated with a second methyl group (ii) a nucleic acid polymerase, (iii) one or more primers that are complementary to a portion of the target nucleic acid, and (iv) a second methyl specific restriction enzyme that is capable of being deactivated and that selectively digests nucleic acids comprising nucleotides that are methylated with the second methyl group, thereby producing a combination; e) maintaining the combination of e) under conditions in which the amplicons of the non target nucleic acid are digested by the second methyl specific restriction enzyme prior to amplification of the genomic DNA that includes the target nucleic acid; and f) amplifying the genomic DNA that includes the target nucleic acid, thereby producing amplicons of the target nucleic acid having one or more nucleotides that are methylated with the first methyl group and the second methyl group, wherein the amplicons of the non target nucleic acid are removed from the sample comprising the genomic DNA that includes the target nucleic acid to be serially amplified. |
