Amplification blocker comprising intercalating nucleic acids (INA) containing intercalating pseudonucleotides (IPN)
Inventors
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-7803580-B2
Publication Date
2010-09-28
Expiration Date
Abstract
An amplification blocker comprising an intercalating nucleic acid (INA) containing two or more internal intercalating pseudonucleotides (IPNs) capable of blocking or reducing nucleic acid amplification. Use of the amplification blocker to block or reduce nucleic acid amplification.
Core Innovation
The disclosed approach provides a method for detecting methylation status of a target region of a nucleic acid molecule by treating the nucleic acid with an agent which modifies cytosine bases but does not modify 5-methyl-cytosine bases under conditions to form a modified nucleic acid template. An amplification reaction is then carried out in which amplification is controlled by intercalating nucleic acid (INA) amplification blockers containing two or more internal intercalating pseudonucleotides.
The blockers are complementary to regions on the modified nucleic acid template and are used together with primers to yield an amplification product or no amplification product depending on methylation status. The internal intercalating pseudonucleotides are essentially planar polyaromatic or heteropolyaromatic compounds capable of co-stacking with nucleobases in a nucleic acid duplex and are not peptide nucleic acids.
The internal intercalating pseudonucleotides prevent extension of at least one of the blockers by a polymerase when the blocker is bound to a nucleic acid template. The disclosed framework also supports detection of conversion status using the same modified template and amplification reaction, and using methylation status indicative of a disease state versus not indicative of a disease state.
Claims Coverage
The document includes multiple independent claims covering methylation status detection, conversion status detection, and disease-state related methylation status. Across the independent claims, the core inventive features are repeated: cytosine base modification without modifying 5-methyl-cytosine, INA amplification blockers with two or more internal intercalating pseudonucleotides, primer placement relative to blocker regions, and amplification results determined by methylation or conversion status, with the further requirement that the internal intercalating pseudonucleotides prevent polymerase extension of at least one bound blocker.
Cytosine modification without 5-methyl-cytosine and modified nucleic acid template
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.
INA amplification blockers with internal intercalating pseudonucleotides to suppress amplification
Providing amplification blockers comprising an intercalating nucleic acid (INA) containing two or more internal intercalating pseudonucleotides, complementary to defined regions on the modified nucleic acid template, wherein the amplification reaction produces an amplification product or no amplification product depending on the target region methylation or conversion status.
Primer regions upstream of target and blocker regions to drive methylation- or conversion-dependent amplification
Providing a first primer complementary to a nucleic acid region upstream from the target region on a first strand of the modified nucleic acid template and a second primer complementary to a nucleic acid region upstream from the second blocker region on a 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.
Planar polyaromatic/heteropolyaromatic internal pseudonucleotides that prevent polymerase extension of bound blockers
The internal intercalating pseudonucleotides are essentially planar polyaromatic or heteropolyaromatic compounds capable of co-stacking with nucleobases in a nucleic acid duplex and are not peptide nucleic acids, and they prevent extension of at least one of the blockers by a polymerase when bound to a nucleic acid template.
Disease-state related methylation status decision
Carrying out an amplification reaction 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.
Across the independent claims, the coverage centers on methylation-status or conversion-status detection using a cytosine-modified template and INA amplification blockers containing two or more internal intercalating pseudonucleotides. The internal pseudonucleotides are planar polyaromatic/heteropolyaromatic co-stacking units that prevent polymerase extension of bound blockers, producing amplification outcomes that depend on whether the target region is methylated/unmethylated or converted/unconverted, including formats that map methylation status to disease-state indication.
Stated Advantages
Enables methylation status detection by yielding an amplification product for one methylation state and no amplification product for the other methylation state.
Enables conversion status detection by yielding an amplification product for converted target regions and no amplification product for unconverted target regions.
Prevents extension of at least one bound amplification blocker by a polymerase via internal intercalating pseudonucleotides.
Supports using methylation status indicative of a disease state versus not indicative of a disease state to determine amplification outcomes.
Documented Applications
Detection of methylation status of a target region of a nucleic acid molecule.
Detection of conversion status of a target region of a nucleic acid molecule.
Detection of methylation status indicative of a disease state.
Interested in licensing this patent?