Products and processes for nucleic acid detection and quantification
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Publication Number
US-12188083-B2
Publication Date
2025-01-07
Expiration Date
Abstract
Provided herein are products and processes for primer extension reactions using chain terminating reagents and enzymes having 3′ to 5′ exonuclease activity, whereby unextended oligonucleotides are digested and extended oligonucleotides are not digested. Also provided herein are products and processes for the detection of the presence, absence or amount of one or more variants of a target nucleic acid species or a plurality of target nucleic acid species comprising primer extension reactions using chain terminating reagents and enzymes having 3′ to 5′ exonuclease activity, whereby unextended oligonucleotides are digested and extended oligonucleotides are not digested.
Core Innovation
The invention provides a method for detecting the presence, absence or amount of variants of one or more target nucleic acid species or amplicons thereof in a sample. Primer oligonucleotides hybridize to variant-discriminating single-base positions at a position 5 to a single base position that differs between variants, generating hybridized oligonucleotides.
The hybridized oligonucleotides are extended up to, or through, the nucleotide position that is different between the variants using an extension composition comprising one or more chain terminating reagents specific for a variant. This generates extended primer oligonucleotides comprising a chain terminating reagent specific for one or more variants of the corresponding target nucleic acid species or amplicon.
The products of extension are contacted with an enzyme having 3 to 5 exonuclease activity, whereby unextended primer oligonucleotides are digested and extended primer oligonucleotides comprising a chain terminating reagent are not digested. The presence, absence or amount of the extended primer oligonucleotides is then analyzed without selective capture from the mixture.
Embodiments include multiplexing, variant categorization for low-abundance variants versus high-abundance variants, reduced concentration of high-abundance terminators, and detection using mass spectrometry, including MALDI-TOF. The disclosed embodiments also include optional prior processing for terminal phosphate removal before extension and kit embodiments comprising the 3 to 5 exonuclease enzyme together with chain terminating reagents.
Claims Coverage
The consolidated claim coverage identifies one independent claim with four core inventive features, together with dependent refinements directed to enzyme identity, label constraints, detection modality, and exclusion of selective capture or solid phase binding.
Variant-discriminating primer hybridization
Primer oligonucleotides hybridize to variants at a position 5 to a single base position that differs between variants, generating hybridized oligonucleotides.
Variant-specific chain terminating primer extension
The hybridized oligonucleotides are extended using an extension composition comprising one or more chain terminating reagents specific for a variant, generating extended primer oligonucleotides comprising a chain terminating reagent.
3 to 5 exonuclease digestion of unextended primers
An enzyme having 3 to 5 exonuclease activity digests unextended primer oligonucleotides while extended primer oligonucleotides comprising a chain terminating reagent are not digested.
Detection of extended primers without selective capture
The presence, absence or amount of the extended primer oligonucleotides is detected without selective capture of the extended primer oligonucleotides from the mixture.
Detecting variants using TTHB178
The enzyme having 3 to 5 exonuclease activity is identified as TTHB178.
Avoidance of detectable labels
The primer oligonucleotides and the chain terminating reagents lack detectable labels attached thereto.
Mass spectrometry analysis
The extended primer oligonucleotides are analyzed by mass spectrometry.
No solid-phase binding or capture
The target nucleic acid species or its amplicons, the primer oligonucleotides, and the products are not captured by binding to a solid phase.
The consolidated inventive coverage combines variant-discriminating primer hybridization, extension with variant-specific chain terminating reagents, selective removal of unextended primers by 3 to 5 exonuclease activity, and detection of the extended primer oligonucleotides without selective capture.
Stated Advantages
Improved signal-to-noise ratio from exonuclease digestion of unextended primers.
Improved signal-to-noise for low-abundance variants.
Variant skewing using reduced concentration of high-abundance terminators.
Multiplexing capability.
Detection by mass spectrometry, including MALDI-TOF.
Documented Applications
Detecting the presence, absence or amount of variants of one or more target nucleic acid species or amplicons thereof in a sample.
Detection and quantification of low-frequency EGFR/KRAS/IDH2/ABL1/ALK/BRAF/Egfr variants in ultra-high sensitivity formats.
Mass spectrometry readout of extended primer oligonucleotides using label/tag concepts (e.g., MALDI-TOF / SpectroCHIP/MassARRAY).
Conditioning approaches compared across bead-based processing versus exonuclease conditioning in iPLEX HS and UltraSEEK ultra-high sensitivity formats.
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