Multiplexed method for the identification and quantitation of minor alleles and polymorphisms

Inventors

Nygren, Anders Olof Herman

Assignees

Agena Bioscience Inc

Abstract

Provided herein are products and processes for detecting the presence or absence of minor nucleic acid species in a sample containing a mixture of minor nucleic acid species and one or more major nucleic acid species, where the amount (frequency or copy number) of the minor nucleic acid species is less than that of the major nucleic acid species. Certain methods include amplifying the mixture and extending the resulting amplicons using chain terminating reagents and extension primers that specifically hybridize to the amplicons, where a chain terminating reagent specific for the major nucleic acid species has a concentration that is less than a chain terminating reagent that is specific for a minor nucleic acid species. Skewing the concentrations of the chain terminating reagents in favor of high concentrations of the chain terminating reagents specific for the minor nucleic acid species relative to a chain terminating reagent specific for a major nucleic acid species improves the detection limit (sensitivity) of detecting minor nucleic acid species present at low frequency or copy number in the mixture. In addition, the signals generated from the extension product of the major nucleic acid species amplicon can serve as a positive control and permit quantification of the minor nucleic acid species relative to the major nucleic acid species in the mixture.

Core Innovation

The invention provides a method for quantifying one or more minor nucleic acid species in a nucleic acid population that also contains a major nucleic acid species, where the minor species are variants of the same major species and are present at a copy number less than that of the major nucleic acid species. The method amplifies target regions to generate an amplified mixture containing major and minor nucleic acid species, and the amplified mixture is used in an extension step that produces chain terminated extension products corresponding to the minor and major nucleic acid species.

During extension, extension primers are contacted under extension conditions that include chain terminating reagents specific for each minor nucleic acid species and for the major nucleic acid species. The primers are extended up to nucleotide positions that are different in the minor nucleic acid species relative to the major nucleic acid species, and the concentrations of the chain terminating reagents are known, with the concentration of the major-specific chain terminating reagent less than the concentration(s) of the minor-specific chain terminating reagent(s).

The method determines the ratio of the amounts of extension products corresponding to each minor nucleic acid species relative to the amount of extension product corresponding to the major nucleic acid species. Based on the ratio and the known concentrations of the chain terminating reagents, the method quantifies the amount(s) of minor nucleic acid species relative to the amount of the major nucleic acid species, and the major nucleic acid species extension product serves as an internal positive control for assay integrity.

Claims Coverage

One independent claim defines a multi-step quantification method using target amplification, allele-specific chain-terminator extension products, ratio determination, and concentration-normalized quantification. Dependent claims further refine copy-number and concentration relationships, extension specificity, terminator type, and labeled detection.

The core inventive concept is allele-specific extension that generates chain terminated extension products, combined with known chain-terminating reagent concentrations that are skewed so the major-specific terminator is at a lower concentration than the minor-specific terminators. The method quantifies minor-to-major amounts using measured ratios of extension products together with the reagent concentration relationships.

Stated Advantages

Documented Applications