US 11,807,848 B2
High throughput discovery of new genes from complex mixtures of environmental microbes
Vadim Beilinson, Durham, NC (US); Janice Jones, Apex, NC (US); Jessica Parks, Raleigh, NC (US); Rebecca E. Thayer, Morrisville, NC (US); Daniel J. Tomso, Bahama, NC (US); Scott Joseph Uknes, Apex, NC (US); Sandy Volrath, Durham, NC (US); and Eric Russell Ward, Durham, NC (US)
Assigned to AgBiome, Inc., Durham, NC (US)
Filed by AGBIOME, INC., Durham, NC (US)
Filed on Jan. 13, 2021, as Appl. No. 17/148,210.
Application 17/148,210 is a continuation of application No. 15/862,184, filed on Jan. 4, 2018, granted, now 10,920,218.
Application 15/862,184 is a continuation of application No. 14/592,473, filed on Jan. 8, 2015, granted, now 9,896,686, issued on Feb. 20, 2018.
Claims priority of provisional application 61/925,422, filed on Jan. 9, 2014.
Prior Publication US 2021/0130811 A1, May 6, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6869 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6827 (2018.01); C12N 15/10 (2006.01)
CPC C12N 15/1065 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6827 (2013.01); C12Q 1/6869 (2013.01)] 23 Claims
 
1. A method for identifying a variant of a gene of interest having less than 95% identity to said gene of interest, in a complex sample, said method comprising:
a) preparing DNA from a complex sample comprising a variant of a gene of interest for hybridization thereby forming a prepared sample DNA, the prepared sample DNA comprising said variant of said gene of interest, wherein said gene of interest comprises a plant pest resistance gene of interest, a plant disease resistance gene of interest, and/or a herbicide tolerance gene of interest;
b) mixing said prepared sample DNA with a labeled bait pool comprising polynucleotide sequences complementary to said plant pest resistance gene of interest, plant disease resistance gene of interest, and/or herbicide tolerance gene of interest;
c) hybridizing the prepared sample DNA to said labeled bait pool under conditions that allow for hybridization of a labeled bait in said labeled bait pool with said variant of said plant pest resistance gene of interest, plant disease resistance gene of interest, and/or herbicide tolerance gene of interest to form one or more hybridization complexes,
wherein said variant of said plant pest resistance gene of interest, plant disease resistance gene of interest, and/or herbicide tolerance gene of interest in the hybridization complexes comprises captured DNA;
d) sequencing said captured DNA to determine a sequence read of said variant of said plant pest resistance gene of interest, plant disease resistance gene of interest, and/or herbicide tolerance gene of interest; and
e) aligning said sequence read to a database of known sequences using a sequence alignment program in order to identify said variant of said plant pest resistance gene of interest, plant disease resistance gene of interest, and/or herbicide tolerance gene of interest having less than 95% identity to said plant pest resistance gene of interest, plant disease resistance gene of interest, and/or herbicide tolerance gene of interest.