Multiplexed KRAS mutation detection assay
Inventors
Oldham-Haltom, Rebecca • Allawi, Hatim • Zou, Hongzhi • Domanico, Michael J. • Lidgard, Graham P.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12060620-B2
Publication Date
2024-08-13
Expiration Date
Abstract
Provided herein is reagent mixture comprising multiplexed amplification reagents and flap assay reagents for detecting, in a single reaction, mutant copies of the KRAS gene that contain any of the 34A, 34C, 34T, 35A, 35C, 35T or 38A point mutations. Methods that employ the reagent mix and kits for performing the same are also provided.
Core Innovation
The invention provides a reagent mixture for amplifying and detecting mutant copies of a KRAS gene that contain specific point mutations. The mixture includes amplification reagents comprising a thermostable polymerase, nucleotides, a set of at least seven different forward primers, and a reverse primer, where each forward primer selectively amplifies a different KRAS mutant allele and the 3′ terminal nucleotide of each forward primer base-pairs with a corresponding mutation position selected from 34A, 34C, 34T, 35A, 35C, 35T and 38A.
The amplification is combined with flap assay reagents comprising a flap endonuclease, a first FRET cassette and a second FRET cassette that produce distinguishable fluorescent signals when cleaved. The reagent mixture further includes a set of at least seven different flap oligonucleotides, where each flap oligonucleotide comprises a nucleotide that base pairs with one of the point mutations, and at least one flap oligonucleotide includes a flap sequence that hybridizes to the first FRET cassette while the remainder hybridize to the second FRET cassette.
The mixture is characterized by its ability to amplify and detect mutant KRAS gene copies in the presence of wild type copies under thermocycled conditions. The nucleic acid sample comprises at least a 100-fold excess of wild type copies of the KRAS gene relative to mutant copies containing the specified point mutations, enabling mutation-dependent fluorescence distinction despite abundant wild type background.
In the disclosed assay context, the approach is directed to multiplexed KRAS mutation detection and real-time fluorescence discrimination based on flap cleavage. An internal control option is described using β-actin with a distinguishable additional FRET channel, and the document describes use for clinical cancer diagnostics and for Noonan/cardio-facio-cutaneous syndrome diagnostics.
Claims Coverage
The provided content includes two independent claims, a reagent mixture and a kit. Each independent claim contains two core inventive groupings: KRAS allele-selective amplification using at least seven forward primers with mutation-specific 3′ mismatches, and flap/FRET detection using first and second FRET cassettes that provide distinguishable fluorescent signals upon cleavage.
Allele-selective KRAS amplification using thermostable polymerase and at least seven forward primers
The 3′ terminal nucleotide of each forward primer base-pairs with a different point mutation in the KRAS gene selected from 34A, 34C, 34T, 35A, 35C, 35T and 38A; each forward primer is fully complementary except for a single base mismatch within 6 bases of the 3′ terminal nucleotide; and each forward primer in combination with the reverse primer selectively amplifies a different KRAS allele defined by the point mutation to which the 3′ terminal nucleotide base-pairs.
Flap/FRET detection with distinguishable first and second FRET cassettes and at least seven flap oligonucleotides
Flap assay reagents comprise a flap endonuclease, a first FRET cassette and a second FRET cassette, wherein the first and second FRET cassettes produce distinguishable fluorescent signals when cleaved; a set of at least seven different flap oligonucleotides each comprising a nucleotide that base pairs with one of the point mutations; and at least one flap oligonucleotide comprises a flap sequence that hybridizes to the first FRET cassette while the remainder hybridize to the second FRET cassette.
Quantified detection characterization under wild-type excess and thermocycled conditions
The reagent mixture is characterized in that, when combined with a nucleic acid sample comprising at least a 100-fold excess of wild type copies of the KRAS gene relative to mutant copies containing one of the point mutations and thermocycled, the reagent mixture can amplify and detect the presence of the mutant copies of the KRAS gene in the sample.
Kit implementation of allele-selective KRAS amplification and flap/FRET detection
A kit comprises amplification reagents with a thermostable polymerase, nucleotides, at least seven different forward primers, and a reverse primer configured such that each forward primer base-pairs with a different KRAS point mutation selected from 34A, 34C, 34T, 35A, 35C, 35T and 38A; each forward primer includes a single base mismatch within 6 bases of the 3′ terminal nucleotide; and each forward primer with the reverse primer selectively amplifies a different KRAS allele defined by the point mutation to which the 3′ terminal nucleotide base-pairs, together with flap assay reagents comprising a flap endonuclease, a first FRET cassette and a second FRET cassette with distinguishable fluorescent signals upon cleavage and a corresponding set of at least seven different flap oligonucleotides each base pairing with one of the point mutations.
Across the independent claims, the key inventive concept is multiplexing allele-selective KRAS point-mutation discrimination using at least seven forward primers with 3′ mutation-defined mismatch positioning, coupled to flap endonuclease-driven FRET detection with a first and second FRET cassette producing distinguishable fluorescent signals, including a wild-type excess characterization enabling detection upon thermocycling.
Stated Advantages
Detects mutant copies of the KRAS gene in a nucleic acid sample containing at least a 100-fold excess of wild type KRAS copies under thermocycled conditions.
Produces distinguishable fluorescent signals from cleavage of first and second FRET cassettes for mutation-dependent detection.
Documented Applications
Clinical cancer diagnostics using the disclosed multiplexed KRAS mutation detection approach.
Noonan/cardio-facio-cutaneous syndrome diagnostics using the disclosed KRAS mutation detection approach.
Detection/correlation in colorectal tissue and stool DNA samples, with real-time discrimination discussed in the document.
Interested in licensing this patent?