SYSTEM
The present disclosure describes technologies that permit sensitive detection of nucleic acids of interest (i.e., nucleic acids whose nucleotide sequence is or includes a target sequence).
1 . A system comprising:
a plurality of nucleic acid molecules having different nucleotide sequences;
a set of ligation oligonucleotides, comprising:
a first ligation oligonucleotide whose nucleotide sequence includes a templating element and a first target hybridization element; and
a second ligation oligonucleotide whose nucleotide sequence includes a second target hybridization element and a Cas recognition element; and
optionally one or more bridging oligonucleotides whose nucleotide sequence is or comprises one or more additional target hybridization elements,
wherein the target hybridization elements bind to different portions of a common target site, so that, when the plurality of nucleic acid molecules includes at least one nucleic acid molecule whose nucleotide sequence includes the target site, then the set of ligation oligonucleotides hybridizes to the target site and forms a gapped nucleic acid strand susceptible to ligation with a ligase to generate a ligated strand.
2 . The system of claim 1 , further comprising a ligase.
3 . The system of claim 1 , wherein one or more of the oligonucleotides is associated with a solid support.
4 . The system of claim 1 , wherein nucleic acids of the plurality are associated with a solid support.
5 . The system of claim 1 , further comprising a second set of ligation oligonucleotides directed to a second target site, different from that of the first set of ligation oligonucleotides.
6 . The system of claim 5 , wherein the templating elements of the first and second sets of ligation oligonucleotides are the same.
7 . The system of claim 5 , wherein the templating elements of the first and second sets of ligation oligonucleotides are different.
8 . The system of any one of claims 5 - 7 , wherein the Cas recognition elements of the first and second sets of ligation oligonucleotides are the same.
9 . The system of any one of claims 5 - 7 , wherein the Cas recognition elements of the first and second sets of ligation oligonucleotides are different.
10 . The system of claim 1 , wherein the templating element is or comprises a promoter or complement thereof
11 . The system of claim 1 , wherein the templating element is or comprises an origin of replication or complement thereof
12 . The system of claim 1 , wherein the templating element is or comprises a binding site for a first extendable primer.
13 . The system of claim 12 , further comprising a first extendable primer.
14 . The system of claim 12 or claim 13 , further comprising a DNA polymerase.
15 . The system of claim 12 or claim 13 , wherein the second ligation oligonucleotide has a sequence that includes the complement of a binding site for a second extendable primer.
16 . The system of claim 15 , further comprising the second extendable primer.
17 . The system of claim 16 , further comprising a DNA polymerase.
18 . The system of claim 17 , wherein the DNA polymerase is thermostable.
19 . The system of any one of the prior claims, further comprising an RNA polymerase.
20 . A set of ligation oligonucleotides, comprising:
a first ligation oligonucleotide whose nucleotide sequence includes an templating element and a first target hybridization element; and
a second ligation oligonucleotide whose nucleotide sequence includes a second target hybridization element and a Cas recognition element; and
optionally one or more bridging oligonucleotides whose nucleotide sequence is or comprises one or more additional target hybridization elements,
wherein the target hybridization elements bind to different portions of a common target site, so that, when the set of ligation oligonucleotides is contacted with a nucleic acid molecule whose nucleotide sequence includes the target site, then the set of ligation oligonucleotides hybridizes to the target site and forms a gapped nucleic acid strand susceptible to ligation with a ligase to generate a ligated strand.
21 . A method comprising steps of:
contacting the set of ligation oligonucleotides of claim 20 with a nucleic acid sample under conditions that permit simultaneous hybridization of the oligonucleotides in the set of ligation oligonucleotides to one or more individual nucleic acid molecules in the nucleic acid sample;
simultaneously or subsequently contacting the set of ligation oligonucleotides with ligase so that the ligated strand is generated if the nucleic acid sample includes at least one nucleic acid molecule whose nucleotide sequence includes the target site.
22 . The method of claim 21 , wherein the templating element is or comprises a binding site for an extendible primer and the method further comprises steps of:
hybridizing the extendible primer to the ligated strand; and
extending the extendible primer so that a duplex is formed.
23 . The method of claim 22 , wherein the templating element further is or comprises a promoter and the method comprises a step of:
transcribing from the promoter so that a transcript comprising the Cas recognition element is generated.
24 . The method of claim 22 , further comprising a step of duplicating one or both strands of the duplex, so that a population of single or double-stranded nucleic acid molecules, each including the Cas recognition element, is generated.
25 . The method of claim 23 or claim 24 , further comprising a step of contacting the Cas recognition element with:
a Cas enzyme characterized by collateral activity; and
a guide RNA that binds to the Cas recognition elements, the contacting being performed while a nucleic acid probe susceptible to collateral cleavage by the Cas enzyme in present; and
detecting cleavage of the nucleic acid probe.
26 . The method of claim 25 , wherein the detecting indicates that a target nucleic acid, whose nucleotide sequence includes the target site, was present in the nucleic acid sample, and the method further comprises:
quantifying the target nucleic amount present in the nucleic acid sample