US 12,391,983 B2
Polynucleotide enrichment and amplification using CRISPR-Cas or Argonaute systems
Jeffrey G. Mandell, San Diego, CA (US); and Molly He, San Diego, CA (US)
Assigned to Illumina, Inc., San Diego, CA (US)
Filed by Illumina, Inc., San Diego, CA (US)
Filed on Dec. 9, 2022, as Appl. No. 18/063,865.
Application 18/063,865 is a continuation of application No. 16/099,953, granted, now 11,542,544, previously published as PCT/US2017/032021, filed on May 10, 2017.
Claims priority of provisional application 62/335,012, filed on May 11, 2016.
Prior Publication US 2023/0295701 A1, Sep. 21, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6844 (2018.01); C12Q 1/686 (2018.01)
CPC C12Q 1/6844 (2013.01) [C12Q 1/686 (2013.01)] 18 Claims
 
1. A method for preparing a library of nucleic acids, comprising:
(i) tagmenting a double-stranded target nucleic acid to obtain a plurality of double-stranded fragments, wherein a first strand of each double-stranded fragment contains a universal sequence; and (ii) amplifying the plurality of double-stranded fragments, comprising: (a) providing a system comprising: a 5′ phosphorylated single-stranded nucleic acid, and an Argonaute protein, wherein the 5′ phosphorylated single-stranded nucleic acid comprises a sequence complementary to a region of the universal sequence,
(b) contacting the plurality of double-stranded fragments with the system to form a plurality of complexes, wherein each complex comprises the 5′ phosphorylated single-stranded nucleic acid hybridized to the first strand, and a displaced region of a second strand of a double-stranded fragment of the plurality of double-stranded fragments, wherein the displaced region comprises a loop,
(c) hybridizing a primer to the displaced region, wherein the primer comprises a sequence complementary to the displaced region, and
(d) extending the hybridized primer with a polymerase.