Patent ID: 7803580
Filing Date: 2010-09-28
Classification: C12Q

Abstract:
1. A method for detecting the methylation status of a target region of a nucleic acid molecule comprising: treating the nucleic acid molecule with an agent which modifies cytosine bases but does not modify 5-methyl-cytosine bases under conditions to form a modified nucleic acid template; providing to an amplification reaction a first amplification blocker comprising an intercalating nucleic acid (INA) containing two or more internal intercalating pseudonucleotides, the first blocker being complementary to the unmethylated target region on a first strand of the modified nucleic acid template; providing to the amplification reaction a second amplification blocker comprising an intercalating nucleic acid (INA) containing two or more internal intercalating pseudonucleotides, the second blocker being complementary to a second region on a second strand complementary to the first strand of the modified nucleic acid template, the second region being downstream of the target region of the first strand of the modified nucleic acid template; providing a first primer complementary to a nucleic acid region upstream from the target region on the first strand of the modified nucleic acid template; providing a second primer complementary to a nucleic acid region upstream from the second blocker region on the second strand complementary to the first strand of the modified nucleic acid template; and carrying out an amplification reaction on the modified nucleic acid template, wherein if the target region is methylated there will be an amplification product and if the target region is unmethylated there will be no amplification product; wherein the internal intercalating pseudonucleotides are essentially planar polyaromatic or heteropolyaromatic compounds that are capable of co-stacking with nucleobases in a nucleic acid duplex and are not peptide nucleic acids and wherein the internal intercalating pseudonucleotides prevent extension of at least one of the blockers by a polymerase when said at least one of the blockers is bound to a nucleic acid template.