Detection of sequences uniquely associated with a dna target region
AssigneePioneer Hi-Bred International, Inc.
InventorsAnanta Acharya, Amanda Gannon, Hena Guo, Kevin Hayes, Robyn Lynn Laskowski, Robert Ebow McEwan, Lasantha Ubayasena, Gina Marie Zastrow-Hayes
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Abstract
The disclosed embodiments concern methods for determining sequences of interest using targeted unique molecular index (TUMI) sequences that are uniquely associated with individual polynucleotide fragments in plants, such as those present in a transgenic event, a site-specific mutation or a wild type variant. System, apparatus, and computer program products are also provided for determining a sequence of interest implementing the methods disclosed.
Claims16
A method for detecting a target nucleotide region of interest in a genome 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 sequence 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 genome based on the sequence information of the polynucleotide sequence uniquely associated with the target nucleotide region of interest.
Dependencies are inferred from the claim text.