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AssigneesWageningen Universiteit, Mscls B.V.
InventorsJohn Van Der Oost, Isabelle Anna Zink, Daniël Christianus Swarts, Max Jan Van Min
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Nucleotide sequences of interest, which may be as yet unknown sequences, comprised in a biological sample may often be present in diminishingly small amounts, meaning there are difficulties in detecting, sequencing and identifying these sequences. A method of enrichment of the sequences of interest prior to sequencing of a sample overcomes the problem. In such a method a library of nucleic acid guides is generated from a portion of the sample itself, and the guides are used with a guide-dependent endonuclease such as an Argonaute, usually a prokaryotic Argonaute (pAgo) in a reaction which cleaves the nucleic acids recognised by the guides in another portion of the same sample, but which spares the low abundance sequences for which no guides have been generated. In this way a sample enriched for rarer or low abundance sequences is provided and used in subsequent steps of detection of sequences present, including sequencing of the enriched sample. The method has wide range of application in scientific research of all kinds and forensics where is it necessary to detect the presence of nucleotide sequences and where needed to allow for the detection and/or sequencing of these.
A method for screening for and/or identifying a nucleotide sequence of interest comprised in nucleic acids of a biological sample, comprising contacting at least a portion of the nucleic acids of the sample with a library of nucleic acid guides and a guide-dependent endonuclease, wherein the library of guides is obtained or derived from at least another portion of the same or a different sample, wherein the sequences of the guides align substantially without mismatches with the entirety of, or at least a portion of, nucleotide sequences that are expected to be present in the sample, wherein sample nucleic acid-endonuclease-guide complexes are formed and have endonuclease activity, and whereby expected nucleic acids in the sample are cleaved, and nucleic acid sequences in the sample which are not sufficiently complementary to any guide sequences are not cleaved.
A method as claimed in any preceding claim, wherein the source of the sample is selected from an organism, a cell culture or an environmental sample.
A method as claimed in any preceding claim, wherein the guides are prepared from a sample of nucleic acid from a first source, and wherein the sample or portion thereof contacted with (a) guides and a guided endonuclease, or (b) endonuclease-guide complexes, is from a sample of nucleic acid from a second source.
A method as claimed in any preceding claim, wherein the guides are prepared from at least a portion of the or another sample by (i) fragmenting sample nucleic acids, (ii) taking a portion of the fragmented nucleic acids, (iii) hybridizing the portion of fragmented nucleic acids to a set of reference probes, wherein the reference probes are optionally shorter than the fragmented nucleic acids, (iv) digesting unhybridized single stranded nucleic acids to form double stranded nucleic acid fragment:probe hybrids, and (v) dissociating the double stranded hybrids so that the digested probes provide the single stranded guides.
A method as claimed in any preceding claim, wherein the guides consist of a multiplicity of sets of nucleic acid guides, and wherein separate portions of the sample are contacted with respective sets of nucleic acid guide-nuclease complexes.
A method as claimed in any preceding claim, wherein at least some of the sequences of the guides are known;
A method as claimed in any preceding claim, wherein at least some of the sequences of the sample nucleic acids are known.
A method as claimed in any preceding claim, wherein guide-endonuclease complexes are formed first by contacting the nucleic acid guide fragments and endonuclease, and then the guide-endonuclease complexes are contacted with the sample.
A method as claimed in any preceding claim, wherein the guides are 5′ phosphorylated.
A method as claimed in any preceding claim, wherein the guides are targeted to a selected region or regions of a genome.
A method as claimed in any preceding claim, wherein the guides are of uniform length, optionally selected from a single length in the range of 8mers-50mers.
A method as claimed in any preceding claim, wherein the guides are DNA, and/or the sample comprises DNA.
A method as claimed in any preceding claim, wherein the nuclease is an Argonaute, preferably a prokaryotic Argonaute (pAgo);
A method as claimed in any preceding claim, further comprising the step of contacting nucleic acids from the sample with a library of guided nucleic acid-binding proteins that do not possess nuclease activity and which comprise a label or tag, wherein the protein-guide complexes bind to, but do not cleave respective nucleic acids which are other than a nucleotide sequence of interest, and wherein the protein-guide complexes and bound nucleic acids are separated from sample nucleic acids which are not bound to protein-guide complexes.
A method as claimed in any preceding claim, wherein the nucleotide sequence of interest comprises a mutation;
A method as claimed in any preceding claim, wherein prior to contacting the sample nucleic acids with guides and guide-dependent endonucleases, either the guides or the nucleic acids from the biological sample are treated with a reagent that reacts with methylated base positions so that nucleotide sequences comprising methylated base positions are selectively preserved from guide sequence dependent endonuclease cleavage.
A method as claimed in any preceding claim wherein a computer is used in the processing and/or analysis of sequence data.
Dependencies are inferred from the claim text.