Systems and methods for ligation
AssigneesUniversity of Washington, Seattle Children's Research Institute
InventorsLisa FRENKEL, Ingrid BECK, Barry LUTZ, Nuttada PANPRADIST
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Abstract
The present technology provides systems and methods for oligonucleotide ligation assays (“OLA”). In some embodiments, OLA devices are provided that incorporate pathogen detection testing and drug resistance testing into a single device using a lateral flow membrane. The OLA devices can be used at point of car settings to aid a clinician in selecting an appropriate therapeutic regimen for an infected patient.
Claims29
A method for detecting a mutation in a target strand of DNA or RNA, the method comprising:
- amplifying the target strand to form amplicons;
- annealing one or more of the amplicons to one or more of a plurality of probes, including—
- a first test probe having a first nucleotide sequence, and
- a common probe having a second nucleotide sequence;
- and
- ligating at least a portion of the plurality of probes via template dependent ligation based on the amplicons to generate ligated probes, wherein the ligated probes comprise first ligated probes including the first test probe ligated to the common probe;
- and
- detecting the ligated probes, wherein detecting the first ligated probe indicates the target strand included the first nucleotide sequence.
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A method for identifying drug-resistant HIV, comprising:
- amplifying a target strand of RNA or DNA containing HIV genetic information to produce amplicons;
- annealing one or more of the amplicons to one or more of a plurality of probes, the plurality of probes including (1) a wild type probe having a nucleotide sequence comprising a wild type sequence of the target strand, (2) at least one mutant probe having a nucleotide sequence comprising at least one potential mutant sequence of the target strand, the at least one potential mutant sequence including a single nucleotide polymorphism compared to the wild type sequence, and (3) at least one common probe;
- ligating individual common probes to individual wild type probes and/or individual mutated probes to form a plurality of ligated probes each comprising a wild type probe and a common probe or a mutant probe and a common probe, wherein ligation occurs when a wild type probe or a mutant probe is hybridized to a complimentary sequence along the amplicons;
- capturing the ligated probes comprising the wild type probe and the common probe in a first region;
- and
- capturing the ligated probes comprising the mutated probe and the common probe in a second region.
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Dependencies are inferred from the claim text.