US 12,454,722 B2
Multiplexed method for the identification and quantitation of minor alleles and polymorphisms
Anders Olof Herman Nygren, San Diego, CA (US)
Assigned to AGENA BIOSCIENCE, INC., San Diego, CA (US)
Filed by Agena Bioscience, Inc., San Diego, CA (US)
Filed on May 2, 2023, as Appl. No. 18/142,512.
Application 18/142,512 is a continuation of application No. 17/098,993, filed on Nov. 16, 2020, granted, now 11,680,289.
Application 17/098,993 is a continuation of application No. 16/669,311, filed on Oct. 30, 2019, granted, now 10,865,439, issued on Dec. 15, 2020.
Application 16/669,311 is a continuation of application No. 16/255,718, filed on Jan. 23, 2019, granted, now 10,513,728, issued on Dec. 24, 2019.
Application 16/255,718 is a continuation of application No. 15/136,024, filed on Apr. 22, 2016, granted, now 10,233,489, issued on Mar. 19, 2019.
Claims priority of provisional application 62/280,951, filed on Jan. 20, 2016.
Claims priority of provisional application 62/152,697, filed on Apr. 24, 2015.
Prior Publication US 2023/0383340 A1, Nov. 30, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/6858 (2018.01); C12Q 1/686 (2018.01)
CPC C12Q 1/6858 (2013.01) [C12Q 1/6827 (2013.01); C12Q 1/686 (2013.01)] 21 Claims
 
1. A method for identifying the presence or absence of one or more minor nucleic acid species in a sample, comprising:
(a) obtaining nucleic acids from a sample that includes a target nucleic acid, wherein the target nucleic acid has a major nucleic acid species and one or more minor nucleic acid species wherein each minor nucleic acid species is a lower frequency or copy number variant of the major nucleic acid species and is present at a frequency or copy number that is less than about 10% of the frequency or copy number of the major nucleic acid species, wherein “about” means±10% of the indicated value;
(b) contacting the nucleic acids of (a) with one or more extension primers, each of which comprises an oligonucleotide that hybridizes to the target nucleic acid, under extension conditions comprising a chain terminator specific for the major nucleic acid species and one or more chain terminators specific for the one or more minor nucleic acid species, wherein the concentration of the chain terminator specific for the major nucleic acid species is between 1% to 20%, or 0.5% to less than 20%, or 0.1% to 10%, or 0.01% to 10%, or 0.01% to 5% of the concentration of each of the chain terminator(s) specific for the minor nucleic acid species and wherein, if a ddNTP chain terminator is present in the extension conditions, the same dNTP is absent from the extension conditions, whereby the one or more extension primers are extended by the chain terminators, thereby generating chain terminated extension products corresponding to the minor nucleic acid species and/or chain terminated extension products corresponding to the major nucleic acid species; and
(c) analyzing the extension products of (b), thereby identifying the presence or absence of the one or more minor nucleic acid species.