US 12,291,744 B2
Methods for enriching a target polynucleotide in a sample
Matthew Nelson, Lino Lakes, MN (US)
Assigned to NEXGEN CANCER DETECTION LLC, Lino Lakes, MN (US)
Filed by NEXGEN CANCER DETECTION LLC, Lino Lakes, MN (US)
Filed on Aug. 30, 2024, as Appl. No. 18/821,044.
Application 18/821,044 is a continuation of application No. 18/691,325, previously published as PCT/US2022/043169, filed on Sep. 11, 2022.
Claims priority of provisional application 63/243,390, filed on Sep. 13, 2021.
Prior Publication US 2025/0011855 A1, Jan. 9, 2025
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6858 (2018.01); C12Q 1/6886 (2018.01)
CPC C12Q 1/6858 (2013.01) [C12Q 1/6886 (2013.01); C12Q 2537/163 (2013.01); C12Q 2600/156 (2013.01)] 7 Claims
 
1. A method for enriching a double-stranded target polynucleotide in a sample to obtain an amplification product, the method comprising the steps of:
(a) preparing a reaction mixture comprising:
(i) the sample, wherein the sample comprises a double-stranded reference polynucleotide and the double-stranded target polynucleotide, wherein the target polynucleotide comprises a genetic alteration and the reference polynucleotide lacks the genetic alteration, wherein the genetic alteration is a substitution of one or more nucleotides, a deletion of one or more nucleotides, and/or an insertion of one or more nucleotides;
(ii) a DNA polymerase that lacks 5′→3′ nuclease activity and comprises 3′→5′ nuclease activity;
(iii) a blocking oligonucleotide that has a length of 8-20 nucleotides, consists of locked nucleic acids (LNAs) comprising a sugar moiety having a 2′-O conjugated to 4′-C via a methylene group, and is complementary to the reference polynucleotide and not complementary to the target polynucleotide;
(iv) a pair of oligonucleotide primers that flank the genetic alteration,
wherein a forward primer of the primer pair is capable of hybridizing to a first strand of the double-stranded reference polynucleotide and to a first strand of the double-stranded target polynucleotide, and
wherein a reverse primer of the primer pair is capable of hybridizing to a second strand of the double-stranded reference polynucleotide and to a second strand of the double-stranded target polynucleotide,
wherein the forward and reverse primers of the primer pair flank the genetic alteration in the target polynucleotide, and
wherein the forward and reverse primers of the primer pair do not overlap with the sequence complementary to the blocking oligonucleotide;
(b) heating the reaction mixture above a temperature to melt the double stranded reference polynucleotide and the double stranded target polynucleotide;
(c) cooling the reaction mixture to a temperature wherein the blocking oligonucleotide hybridizes to the reference polynucleotide to form a blocking duplex,
but wherein the blocking oligonucleotide does not hybridize to the target polynucleotide, and wherein the forward and reverse primers of the primer pair do not hybridize to the reference polynucleotide or to the target polynucleotide;
(d) cooling the reaction mixture, wherein the forward primer hybridizes to the reference polynucleotide and the reverse primer hybridizes with the reference polynucleotide, and wherein the forward primer hybridizes with the target polynucleotide and the reverse primer hybridizes with the target polynucleotide;
(e) extending the primers hybridized to the target polynucleotide or to the reference polynucleotide; and
(f) repeating steps (b) through (e) for two or more cycles to form the amplification product containing target polynucleotides, thereby enriching the quantity of target polynucleotides relative to the reference polynucleotides in the amplification product as compared to the sample.