Amplification blocker comprising intercalating nucleic acids (INA) containing intercalating pseudonucleotides (IPN)

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 methylated 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 unmethylated there will be an amplification product and if the target region is methylated 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.

A method for detecting a conversion 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 unconverted 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 converted there will be an amplification product and if the target region is unconverted 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.

A method for detecting a conversion 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 converted 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 unconverted there will be an amplification product and if the target region is converted 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.

A method for detecting a 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 a target region on a first strand of the modified nucleic acid template having a methylation status indicative of the presence of a disease state;
• 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 has a methylation status which is not indicative of a disease state there will be an amplification product and if the target region has a methylation status which is indicative of a disease state 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.

A method for detecting a 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 a target region on a first strand of the modified nucleic acid template having a methylation status which is not indicative of a disease state;
• 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 region on the complementary second strand downstream 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;
• carrying out an amplification reaction on the modified nucleic acid template, wherein if the target region has a methylation status indicative of a disease state there will be an amplification product and if the target region has a methylation status which is not indicative of a disease state there will be no amplification product;
• wherein the internal intercalating pseudonucleotides are being 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.

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;
• providing third and fourth amplification blockers comprising an intercalating nucleic acid (INA) containing two or more internal intercalating pseudonucleotides to the amplification reaction, the third and fourth blockers being complementary to regions internal to the first and second blocker regions;
• and
• providing third and fourth primers complementary to regions external to the third and fourth blocker regions and internal to the first and second primer regions;
• 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.

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 methylated 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;
• providing third and fourth amplification blockers comprising an intercalating nucleic acid (INA) containing two or more internal intercalating pseudonucleotides to the amplification reaction, the third and fourth blockers being complementary to regions internal to the first and second blocker regions;
• providing third and fourth primers complementary to regions external to the third and fourth blocker regions and internal to the first and second primer regions;
• and
• carrying out an amplification reaction on the modified nucleic acid template, wherein if the target region is unmethylated there will be an amplification product and if the target region is methylated 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.