Sequencing using non-natural nucleotides
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Publication Number
US-12173365-B2
Publication Date
2024-12-24
Expiration Date
Abstract
The present disclosure provides methods and systems for nucleic acid sequencing. Such systems and methods may achieve context-independent incorporation, have reduced context-dependence or have context-dependence that is amenable to calibration and modeling. Such systems and methods may also reduce misincorporation.
Core Innovation
The invention relates to determining a nucleic acid sequence of a target nucleic acid molecule by repeatedly incorporating nucleotide mixtures into nucleic acid molecules immobilized to a support. The immobilized plurality exhibits sequence homology to the target nucleic acid molecule, and the support is operatively coupled to a detector so that incorporation is monitored through detection signals.
A nucleotide mixture is directed to the immobilized plurality and comprises at least a first subset of nucleotide analogs and a second subset of nucleotide analogs. The nucleotide analogs of the first and second subsets are different from one another, wherein a given one of the first subset comprises a reporter moiety and is detectable by the detector, while a given one of the second subset is not detectable by the detector.
The method incorporates the nucleotide mixture into the target-homologous immobilized nucleic acid molecules, detects the given one of the first subset using the detector, and repeats incorporation and detection to determine the nucleic acid sequence. In an analyzing method, the target nucleic acid molecule immobilized to a support is brought into contact with a nucleotide mixture of first and second sets of nucleotide analogs of the same canonical base type, where only one set but not both is detectable, and detecting one or more signals determines at least a portion of a nucleic acid sequence.
Claims Coverage
Two independent claims are provided, one focused on determining a nucleic acid sequence via a two-subset detectable and non-detectable nucleotide mixture and repeated detection, and one focused on analyzing a target nucleic acid with first and second detectable-only-one-set nucleotide analog subsets of the same canonical base type. The independent claims collectively emphasize detector-linked immobilized homologous nucleic acids, distinguishability by detectability via a reporter moiety, and repetition to obtain sequence information.
Detector-coupled immobilized target-homologous nucleic acids
providing a plurality of nucleic acid molecules immobilized to a support, wherein each of said plurality of nucleic acid molecules exhibits sequence homology to said target nucleic acid molecule, and wherein said support is operatively coupled to a detector
Two subsets of distinguishable detectable and non-detectable nucleotide analogs
directing a nucleotide mixture to said plurality of nucleic acid molecules immobilized to said support, which said nucleotide mixture comprises at least a first subset of nucleotide analogs and a second subset of nucleotide analogs, wherein nucleotide analogs of said first subset and said second subset are different from one another, wherein a given one of said first subset of nucleotide analogs comprises a reporter moiety and is detectable by said detector, and wherein a given one of said second subset of nucleotide analogs is not detectable by said detector
Repeated incorporation and detection of the detectable subset to determine sequence
incorporating said nucleotide mixture comprising at least said first subset of nucleotide analogs and said second subset of nucleotide analogs into said plurality of nucleic acid molecules; using said detector to detect said given one of said first subset of nucleotide analogs; and repeating, thereby determining said nucleic acid sequence of said target nucleic acid molecule
Analyzing with two detectable-only-one-set analog sets of the same canonical base type
bringing said target nucleic acid molecule immobilized to a support in contact with a nucleotide mixture comprising a first set of nucleotide analogs and a second set of nucleotide analogs, wherein nucleotide analogs of said first set and nucleotide analogs of said second set are different nucleotide analogs but of the same canonical base type, and wherein nucleotide analogs of said first set or said second set, but not both, are detectable; and detecting one or more signals from said target nucleic acid molecule, thereby determining at least a portion of a nucleic acid sequence of said target nucleic acid molecule
The claim set is directed to sequencing or analyzing nucleic acids by contacting immobilized, sequence-homologous nucleic acids with nucleotide mixtures that include two different analog subsets or sets engineered so that only the reporter moiety-bearing subset or set is detectable, followed by detector-based signal detection. The independent claims further require repetition for the determination method or signal detection for the analyzing method to obtain sequence information.
Stated Advantages
Documented Applications
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