Orthogonal cleavage ligation sequencing (ocls)
AssigneeOregon Health & Science University
InventorCorey M. Dambacher
Life of this patent
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Abstract
The present disclosure relates to methods and systems involved with spatial encoding/decoding of features. Provided orthogonal cleavage-ligation sequencing (OCLS) systems encode two or more orthogonal recognition sites for restriction enzymes, such as Type IIS enzymes, for decoding of visual barcodes. Decoding employs specific orthogonal ligation of differentially-labeled probes, which enables visual distinguishing of the barcodes at each feature. This process can be repeated over cycles and in parallel.
Claims46
A visual barcode, comprising:
- at least two or more double-stranded DNA (dsDNA) oligonucleotide segments (cassettes) functionally linked linearly to each other, and each comprising within the sequence of the dsDNA segment:
- a recognition site (RS) for a specific restriction endonuclease (RE),
- a designed cleavage site (CS), or
- both a RS and a CS;
- and
- an untethered end at one end of the visual barcode.
An orthogonal cleavage-ligation sequencing (OCLS) barcode comprising two or more dsDNA segments (cassettes), each dsDNA segment containing a recognition site (RS) for a specific restriction endonuclease (RE), and one or more overlapping region(s) configured to permit ligation to flanking dsDNA segments to form a chain of segments, which chain of segments constitutes the OCLS barcode.
A visually detectable orthogonal ligation probe comprising:
- a fully or partially double-stranded DNA oligonucleotide, having a 3′ or 5′ overhang of at least two nucleotides, the sequence of which includes:
- a recognition site (RS) for a specific restriction endonuclease (RE),
- a designed cleavage site (CS), or
- both a RS and a CS;
- and
- covalently attached to the fully or partially dsDNA oligonucleotide, a visually detectable label.
An orthogonal cleavage-ligation sequencing (OCLS) oligonucleotide pair, comprising:
- a visual barcode, comprising:
- at least two or more double-stranded DNA (dsDNA) oligonucleotide segments (cassettes) functionally linked linearly to each other, and each comprising within the sequence of the dsDNA segment:
- a recognition site (RS) for a specific restriction endonuclease (RE),
- a designed cleavage site (CS), or
- both a RS and a CS;
- and
- an untethered end at one end of the visual barcode;
- and
- a visually detectable orthogonal ligation probe, comprising:
- a fully or partially double-stranded DNA oligonucleotide, having a 3′ or 5′ overhang of at least two nucleotides, the sequence of which includes:
- a recognition site (RS) for a specific restriction endonuclease (RE), or
- both a RS and a cleavage site (CS);
- and
- covalently attached to the fully or partially dsDNA oligonucleotide, a visually detectable label;
- wherein cleavage of the RS or the CS in the visual barcode produces a single-stranded “sticky end” overhang having a sequence with full complementary to a sticky end (overhang) produced by cleavage of the RS in the visually detectable orthogonal ligation probe.
A method of encoding a visual barcode, comprising:
- contacting a double-stranded DNA (dsDNA) oligonucleotide tethered at a first end to a solid support, which dsDNA oligonucleotide has a single-stranded overhang at a second, untethered end, with a first dsDNA segment having a first overhanging end compatible for binding to the single-stranded overhang of the tethered dsDNA oligonucleotide and a second overhanging end, and comprising within the sequence of the first dsDNA segment:
- a recognition site (RS) for a specific restriction endonuclease (RE),
- a designed cleavage site (CS), or
- both a RS and a CS;
- which contacting occurs under conditions sufficient to allow specific hybridization of the single-stranded overhang of the first dsDNA segment to the single-stranded overhang of the tethered dsDNA oligonucleotide;
- ligating the first dsDNA segment to the tethered dsDNA oligonucleotide, to form a first captured barcode segment, which comprises the second overhanging end of the first dsDNA segment;
- contacting the first captured barcode segment with a second dsDNA segment having a first overhanging end compatible for binding to the single-stranded overhang of the first captured barcode segment oligonucleotide and a second overhanging end, and comprising within the sequence of the second dsDNA segment:
- a RS for a specific RE different from the RS/RE in the first dsDNA segment,
- a designed CS different from the designed CS in the first dsDNA segment, or both;
- and
- ligating the second dsDNA segment to the first captured barcode segment, to form a second captured barcode segment, which comprises the second overhanging end of the second dsDNA segment, which first captured barcode segment and second captured barcode segment constitute the visual barcode.
A method of decoding a visual barcode, comprising:
- contacting, in a milieu, at least one double-stranded DNA (dsDNA) orthogonal cleavage-ligation sequencing (OCLS) barcode comprising at least one restriction site (RS), with a restriction endonuclease (RE) that recognizes that RS, under conditions sufficient for the RE to cleave the dsDNA OCLS barcode, which RS/RE cleavage results in a single-stranded overhang to produce a partially single-stranded (ss) DNA-partially dsDNA OCLS barcode;
- contacting the partially ssDNA-partially dsDNA OCLS barcode with at least one orthogonal ligation probe comprising a dsDNA oligonucleotide comprising an overhang at a first end and a visually detectable label, under conditions sufficient for the overhang of the orthogonal ligation probe to bind by base pair-mediated hydrogen bonding to the overhang on the partially ssDNA-partially dsDNA OCLS barcode if the sequence of the overhang of the orthogonal ligation probe is the reverse complement of the sequence of the overhang of the partially ssDNA-partially dsDNA OCLS barcode;
- if base pair-mediated binding occurs, ligating the orthogonal ligation probe to the partially single-stranded (ss) DNA-partially dsDNA OCLS barcode to produce a captured probe;
- and
- detecting presence, absence, and/or quantity of captured probe by imaging the visually detectable label.
A method of decoding visual barcodes using orthogonal cleavage-ligation sequencing (OCLS) essentially as described herein.
An improved system for molecular barcoding, comprising repeated cycles of labeling, orthogonal cleavage, ligation, and imaging in order to identify individual features, wherein the orthogonal cleavage comprises cleaving a double-stranded DNA barcode with a Type IIS Restriction Endonuclease.
Orthogonal cleavage-ligation sequencing (OCLS) system, as illustrated in FIGS. 5A-5E.
Dependencies are inferred from the claim text.