US 12,410,470 B2
Detection of sequences uniquely associated with a DNA target region
Ananta Acharya, Johnston, IA (US); Amanda Marie Gannon, Dallas Center, IA (US); Hena Guo, Johnston, IA (US); Kevin Hayes, Urbandale, IA (US); Robyn Lynn Laskowski, Des Moines, IA (US); Robert Ebow McEwan, Ankeny, IA (US); Carisa Townsend, Palmetto, GA (US); Lasantha Ubayasena, West Lafayette, IN (US); and Gina Marie Zastrow-Hayes, Urbandale, IA (US)
Assigned to PIONEER HI-BRED INTERNATIONAL, INC., Johnston, IA (US)
Appl. No. 17/754,890
Filed by PIONEER HI-BRED INTERNATIONAL, INC., Johnston, IA (US)
PCT Filed Oct. 9, 2020, PCT No. PCT/US2020/055079
§ 371(c)(1), (2) Date Apr. 14, 2022,
PCT Pub. No. WO2021/076423, PCT Pub. Date Apr. 22, 2021.
Prior Publication US 2024/0093315 A1, Mar. 21, 2024
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6869 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6851 (2018.01); C12Q 1/6853 (2018.01); C12Q 1/6855 (2018.01); C12Q 1/6858 (2018.01); C12Q 1/686 (2018.01); C12Q 1/6874 (2018.01); C12Q 1/6895 (2018.01)
CPC C12Q 1/6869 (2013.01) [C12Q 1/6806 (2013.01); C12Q 1/6851 (2013.01); C12Q 1/6853 (2013.01); C12Q 1/6855 (2013.01); C12Q 1/6858 (2013.01); C12Q 1/686 (2013.01); C12Q 1/6874 (2013.01); C12Q 1/6895 (2013.01); C12Q 2525/155 (2013.01); C12Q 2525/179 (2013.01); C12Q 2525/185 (2013.01); C12Q 2525/191 (2013.01); C12Q 2537/149 (2013.01); C12Q 2549/00 (2013.01); C12Q 2600/156 (2013.01); C12Q 2600/172 (2013.01)] 21 Claims
 
1. A method for detecting a target nucleotide region of interest in a plant genome, wherein the plant genome is from wheat, potato, cotton, apple, alfalfa or canola, by directional nucleotide sequencing, the method comprising:
providing a first polynucleotide fragment comprising a first polynucleotide PCR primer sequence specific to the target nucleotide region of interest, a sequencing primer binding site sequence, and a polynucleotide sequence region uniquely associated with the target nucleotide region of interest, wherein the sequencing primer binding site is positioned between the polynucleotide sequence uniquely associated with the target nucleotide region of interest and the first polynucleotide PCR primer sequence specific to the target nucleotide region of interest;
providing a second polynucleotide fragment comprising a second polynucleotide PCR primer sequence specific to the target nucleotide region of interest;
performing a PCR to create one or more amplicons comprising the sequencing primer binding site sequence, the polynucleotide sequence uniquely associated with the target nucleotide region of interest, and the target nucleotide region of interest;
sequencing the one or more amplicons using the sequencing primer in a direction away from the target nucleotide region of interest so that the polynucleotide sequence uniquely associated with the target nucleotide region of interest is sequenced and the target nucleotide region of interest in the one or more amplicons is not sequenced; and
detecting the target nucleotide region in the plant genome based on the sequence information of the polynucleotide sequence uniquely associated with the target nucleotide region of interest.