Liquid Sample Workflow for Nanopore Sequencing

Abstract

The present invention relates to a method for characterizing a target DNA polynucleotide using rolling circle amplification (RCA) and a synthetic single-stranded guide RNA (sgRNA) to recognize and cleave a WT version of the target DNA polynucleotide. A characterization step is also provided, which is based on the use of a transmembrane pore and a DNA translocase that controls the movement of the DNA polynucleotide through the transmembrane pore. Further contemplated is a kit comprising one or more oligonucleotides specific for at least a portion of the target DNA polynucleotide, an sgRNA specific for the WT version of the target DNA polynucleotide, and a nucleic acid binding protein guided by the sgRNA.

Description

Technical Field

[0001] The present invention relates to a method for characterizing a target DNA polynucleotide, which uses rolling circle amplification (RCA) and a synthetic single-stranded guide RNA (sgRNA) to recognize and cut the WT version of the target DNA polynucleotide. It is also provided that it is based on the characterization step of using a transmembrane pore and a DNA translocase, which controls the movement of the DNA polynucleotide through the transmembrane pore. Further envisioned is a kit, which includes one or more oligonucleotides specific to at least a portion of the target DNA polynucleotide, an sgRNA specific to the WT version of the target DNA polynucleotide, and a nucleic acid binding protein guided by the sgRNA. Background Art

[0002] Next generation sequencing (NGS) is a major driving force in genetics and molecular research, especially modern diagnostics in the field of cancer medicine. The technology provides a powerful method for studying DNA or RNA samples. New and improved methods and protocols have been developed to support a wide range of applications, including analysis of genetic variation and sample-specific differences. In order to improve the method, methods have been developed that are intended to be targeted and enriched in sequencing libraries by focusing on specific sequences, transcripts, genes or genomic subregions or by eliminating unwanted sequences.

[0003] Targeted enrichment can be useful in many cases, for example, where it is necessary to analyze specific portions of the entire genome. The efficient sequencing of a complete exome (all transcribed sequences) is a typical example of this method. Further examples include enrichment of specific transcripts, enrichment of mutation hot spots, or exclusion of interfering nucleic acid species.

[0004] Current targeted enrichment technologies include (i) hybridization capture, in which nucleic acid strands derived from an input sample are specifically hybridized in solution or on a solid support to pre-prepared DNA fragments complementary to a targeted region of interest, thereby physically capturing and isolating the sequence of interest; (ii) selective circularization or molecular inversion probes (MIPs), in which single-stranded DNA loops including the sequence of the target region are formed in a highly specific manner by gap-filling and ligation chemistry to form structures with common DNA elements, which are then used to selectively amplify the target region of interest; and (iii) polymerase chain reaction (PCR) amplification, in which PCR is directed toward a targeted region of interest by performing multiple long-distance PCRs in parallel, a limited number of standard multiplex PCRs, or highly multiplexed PCR methods, amplifying a very large number of short fragments (Mertes et al., 2011, Briefings infunctional Genomics, 10, 6, 374-386).

[0005] However, in order to utilize these technologies, suitable biopsy materials must first be obtained from the patient, especially if the method is used in cancer diagnosis. Solid tissue biopsies are expensive and in many cases painful for the patient. In addition, solid tissue biopsies cannot always be performed because they do not reflect the current disease dynamics or sensitivity to treatment, such as in the case of cancer. Therefore, it is necessary to provide a method to replace solid tissue biopsies while increasing the sensitivity of the method. An emerging solution to counteract the sensitivity limitations of NGS and the invasiveness of obtaining tissue samples is enriched liquid biopsy (Hesse et al., 2015, Advances in Molecular Diagnostics, 1, 1, 2-7). Liquid biopsy is usually a blood sample, from which circulating cells such as circulating tumor cells (CTC) or circulating cell-free DNA (cfDNA) can be separated. These cell-free DNA (cfDNA) or circulating nucleic acids (including DNA, and RNA species such as micoRNA) remain in the blood for a period of time as circulating fragments, and like other blood analytes, can be evaluated by simple blood sampling. However, cfDNA and similar circulating nucleic acids are challenging analytes because their plasma varies greatly, not only from person to person, but also depending on the disease condition. For example, cfDNA levels in plasma are usually limited to 1 to 100 ng / ml plasma, and in addition, the signal-to-noise ratio between cfDNA fragments and normal cfDNA is low. cfDNA and other circulating nucleic acid fragments are also very small, with an average size of approximately 60-180 bp, requiring specific extraction and NGS library size selection protocols.

[0006] Therefore, there is a need for simplified, cost- and resource-sensitive enrichment and sequencing methods that allow for efficient characterization of target DNA polynucleotides, particularly those derived from liquid biopsy tissues. Summary of the invention

[0007] The present invention meets this need and provides a method for characterizing a target DNA polynucleotide, the method comprising: (i) providing a mixture of DNA polynucleotides, the mixture of DNA polynucleotides comprising at least a wild-type (WT) version and a mutant version of the DNA polynucleotide; (ii) amplifying the mixture of DNA polynucleotides of step (i) by rolling circle amplification (RCA) to provide a library of amplified and concatenated DNA polynucleotides; (iii) recognizing and cutting the WT version of the target DNA polynucleotide by using a synthetic single-stranded guide RNA (sgRNA) specific for the WT version and a nucleic acid binding protein guided by the sgRNA, preferably Cas9; (iv) size-selecting the uncut mutant target DNA polynucleotide; and (v) characterizing the uncut mutant target DNA polynucleotide. Due to the removal of the WT sequence, the method advantageously allows for a reduction in sequencing depth. The method is also suitable for multiplexing different patient samples and allows for enrichment of selected regions or combinations (panels) of genes or exons.

[0008] In a preferred embodiment, step (v) as described above comprises the following sub-steps: (va) connecting an adaptor polynucleotide associated with a DNA translocation enzyme and at least one cholesterol tether segment to the mutant target DNA polynucleotide obtained in step (iv); (vb) contacting the modified DNA polynucleotide obtained in step (va) with a transmembrane pore so that the DNA translocation enzyme controls the movement of the DNA polynucleotide through the transmembrane pore, and the cholesterol tether anchors the DNA polynucleotide near the transmembrane pore; and (vc) performing one or more measurements during the movement of the DNA polynucleotide through the transmembrane pore, wherein the measurement indicates one or more characteristics of the DNA polynucleotide, thereby characterizing the target DNA polynucleotide. Therefore, the long reads (reads) with repetitive sequences obtained by the above method significantly improve the sequencing accuracy for mutation calls in transmembrane pore-based sequencing.

[0009] In another preferred embodiment, the method as described above further comprises a step (ia) of end repair and A-tail addition of the DNA polynucleotide after step (i).

[0010] In another preferred embodiment, the method as described above further comprises, after step (ia), a step (ib) of circularizing the DNA polynucleotide using a stem-loop oligonucleotide, wherein the stem-loop oligonucleotide comprises a barcoding sequence and a restriction enzyme recognition site.

[0011] It is particularly preferred to carry out the rolling circle amplification with one or more oligonucleotides specific for at least a portion of the target DNA polynucleotide. In another specific embodiment of the present invention, the one or more oligonucleotides specific for at least a portion of the target DNA polynucleotide are hexamers, heptamers and / or octamers.

[0012] In another embodiment of the present invention, rolling circle amplification is performed until the amplified DNA polynucleotide has a size of at least about 300 nucleotides. It is particularly preferred that it has a size of at least about 3000 nucleotides.

[0013] In another embodiment, the resulting rolling circle amplification product is repaired using T7 endonuclease, DNA polymerase, and optionally, ligase.

[0014] In another embodiment, the target DNA polynucleotide represents a gene, one or more exons of a gene, an intergenic region, a non-transcribed regulatory region and / or an open reading frame or a sub-portion thereof; or a group of different genes, a group of one or more exons of different genes, a group of intergenic regions, a group of non-transcribed regulatory regions and / or a group of open reading frames or a sub-portion thereof, or any combination of any of the aforementioned elements.

[0015] Preferably, the target DNA polynucleotide is cell-free DNA (cfDNA). Particularly preferably, the cfDNA is derived from a liquid biopsy.

[0016] In a specific embodiment of the method of the invention, the characterization of the DNA polynucleotide is (i) determining the length of the DNA polynucleotide, (ii) determining the identity of the DNA polynucleotide, or (iii) determining the sequence of the DNA polynucleotide. Determining the sequence of the DNA polynucleotide is particularly preferred.

[0017] In a particular embodiment of the method utilizing a transmembrane pore as defined above, the DNA translocase is a DNA helicase, such as a Hel308 helicase, a RecD helicase, a XPD helicase or a Dda helicase.

[0018] In another embodiment of the method, the transmembrane pore is a protein pore derived from hemolysin, leukocidin, MspA, MspB, MspC, MspD, CsgG, lysenin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) or WZA.

[0019] In another aspect, the present invention relates to a kit for characterizing a target DNA polynucleotide, the kit comprising one or more oligonucleotides specific to at least a portion of the target DNA polynucleotide, a synthetic single-stranded guide RNA (sgRNA) specific to the WT version of the target DNA polynucleotide, and a sgRNA-guided nucleic acid binding protein. It is particularly preferred that the sgRNA-guided nucleic acid binding protein is a Cas9 endonuclease.

[0020] In a specific embodiment of the kit, the kit further comprises a DNA translocase and a cholesterol tether.

[0021] It is to be understood that the features mentioned above and those yet to be explained hereinafter can be used not only in the respective combination indicated but also in other combinations or alone, without departing from the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A schematic diagram showing the steps of characterizing a target DNA polynucleotide using rolling circle amplification (RCA) and synthetic single-stranded guide RNA (sgRNA) according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0023] Although the invention will be described with respect to specific embodiments, this description should not be construed in a limiting sense.

[0024] Before describing exemplary embodiments of the present invention in detail, definitions important for understanding the present invention are given.

[0025] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include their respective plural forms as well, unless the context clearly dictates otherwise.

[0026] In the context of the present invention, the terms "about" and "approximately" denote a range of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term generally denotes a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the indicated numerical value.

[0027] It should be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of" or "consisting essentially of" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising at least a certain number of embodiments, this means also including a group that preferably consists only of these embodiments.

[0028] Furthermore, the terms "(i)", "(ii)", "(iii)" or "(a)", "(b)", "(c)", "(d)" or "first", "second", "third", etc. in the description or claims are used to distinguish between similar elements and not necessarily to describe a sequential or chronological order.

[0029] It should be understood that the terms so used are interchangeable where appropriate, and that the embodiments of the invention described herein are capable of operating in other sequences than those described or illustrated herein. If these terms relate to steps of a method, process, or use, there is no temporal or temporal continuity between the steps, i.e., unless otherwise specified, the steps may be performed simultaneously, or there may be a temporal interval of seconds, minutes, hours, days, weeks between such steps.

[0030] It should be understood that the present invention is not limited to the specific methods, protocols, etc. described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0031] The drawings should be considered schematic representations, and the elements shown in the drawings are not necessarily shown to scale. Rather, the various elements are represented so that their function and general purpose are apparent to those skilled in the art.

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0033] As described above, the present invention relates in one aspect to a method for characterizing a target DNA polynucleotide, the method comprising: (i) providing a mixture of DNA polynucleotides, the mixture of DNA polynucleotides comprising at least a wild-type (WT) version and a mutant version of the DNA polynucleotide; (ii) amplifying the mixture of DNA polynucleotides of step (i) by rolling circle amplification (RCA) to provide a library of amplified and concatenated DNA polynucleotides; (iii) recognizing and cutting the WT version of the target DNA polynucleotide by using a synthetic single-stranded guide RNA (sgRNA) specific for the WT version and a nucleic acid binding protein guided by the sgRNA, preferably Cas9; (iv) size-selecting the uncut mutant target DNA polynucleotide; and (v) preferably characterizing the uncut mutant target DNA polynucleotide by sequencing.

[0034] As used herein, the term "target DNA polynucleotide" refers to any DNA molecule suitable for molecular analysis. In a specific embodiment of the present invention, the target polynucleotide represents a gene, one or more exons of a gene, an intergenic region, a non-transcriptional regulatory region and / or an open reading frame or a sub-portion thereof. In other embodiments, the target polynucleotide can also be a group of different genes, one or more exons of a group of different genes, a group of intergenic regions, a group of non-transcriptional regulatory regions and / or a group of open reading frames or a sub-portion thereof. The target DNA polynucleotide can be further provided as a single DNA molecule, or preferably provided in the form of a DNA molecule library, such as representing a gene as described above, one or more exons of a gene, an intergenic region, a non-transcriptional regulatory region and / or an open reading frame or a sub-portion thereof, or a group of different genes, one or more exons of a group of different genes, a group of intergenic regions, a group of non-transcriptional regulatory regions and / or a group of open reading frames or a sub-portion thereof.

[0035] In the first step of the method of the present invention, a mixture of DNA polynucleotides is provided, which mixture of DNA polynucleotides at least includes a wild-type (WT) version and a mutant version of the DNA polynucleotide.

[0036] A "DNA polynucleotide" may be naturally occurring or artificial. It may include modifications, such as oxidized or methylated nucleotides. In certain embodiments, a DNA polynucleotide may also include artificial additives, such as tags or markers.

[0037] The DNA polynucleotide can be of any possible origin, such as prokaryotic, eukaryotic, archaebacterial or viral. The DNA polynucleotide to be characterized according to the present invention can have any known possible biological or cellular function. For example, it can be any naturally occurring or synthetic polynucleotide.

[0038] The provision of DNA polynucleotides can include extraction and / or purification of DNA molecules, separation from cell debris, filtration, elution from a column (e.g., silica gel column), centrifugation, digestion (e.g., RNase digestion) or removal of nucleotides or protein components in a sample. DNA polynucleotides are preferably provided in a buffer solution that includes any suitable composition that prevents DNA degradation. The buffer can, for example, be 3 / 40 buffer or TE buffer (10mM Tris, 1mM EDTA) including EDTA (e.g., 0.1mM). Preferably, the buffer can include a DNA enzyme blocking compound or a DNA enzyme inhibitor. It is also contemplated that a DNA polynucleotide, such as obtained from an RNA polynucleotide by reverse transcription, is provided.

[0039] The provision of DNA polynucleotides can also involve obtaining a sample from a subject and processing it, such as extraction of DNA or a preparatory step to promote DNA extraction. As used herein, the term "sample from a subject" refers to any biological material obtained from a subject by a suitable method known to those skilled in the art. The sample used in the context of the present invention should preferably be collected in a clinically acceptable manner, more preferably in a manner to preserve DNA polynucleotides. Biological samples can include body tissues and / or fluids, such as blood or blood components (such as serum or plasma), sweat, sputum or saliva, semen and urine, and feces or feces samples. Particularly preferably, the sample is a liquid biopsy sample.

[0040] As used herein, the term "liquid biopsy" refers to the sampling and analysis of non-solid biological tissues (mainly blood). Sampling is non-invasive to a large extent, allowing frequent repeated sampling, thereby helping to track mutations or modifications that change over time, or verifying the efficiency of treatment. Liquid biopsy sampling is generally intended to obtain different types of cells and / or nucleic acids. For example, circulating endothelial cells (CEC) or cell-free fetal DNA (cffDNA) can be sampled. Preferably, circulating tumor cells (CTC), particularly cell-free DNA are sampled. Therefore, in a particularly preferred embodiment, the DNA polynucleotide to be analyzed according to the present invention is cell-free DNA.

[0041] In a further embodiment, the biological sample can contain a cell extract derived from an epithelial cell or a cell population comprising an epithelial cell, preferably a neoplastic epithelial cell or an epithelial cell derived from a suspected neoplastic tissue. Alternatively, the biological sample can be derived from the environment, such as from soil, lakes, rivers, etc., or from an animal source.

[0042] In some embodiments, cells can be used as the main source of DNA polynucleotides. Therefore, if necessary, cells can be purified from the body tissues and body fluids obtained, and then further processed to obtain DNA polynucleotides. In some embodiments, particularly after initial treatment, samples can be merged (pool). The present invention preferably envisions the use of non-merged samples.

[0043] In a specific embodiment of the present invention, the content of the biological sample can also be subjected to an enrichment step. For example, the sample can be contacted with a ligand specific to the cell membrane or organelle of certain cell types, for example, functionalized with magnetic particles. The material enriched by the magnetic particles can be subsequently used to extract DNA polynucleotides. In other embodiments of the present invention, a biopsy or excision sample can be obtained and / or used. Such samples can include cells or cell lysates. In addition, cells, such as tumor cells, can be enriched by a filtration process of a fluid or liquid sample (such as blood, urine, sweat, etc.). As described above, such a filtration process can also be combined with an enrichment step based on ligand-specific interactions.

[0044] As used herein, a "mixture" of DNA polynucleotides refers to a situation in which a sample or any starting composition includes at least two (wild type (WT) and mutant versions) of a target DNA polynucleotide. The term "wild type of target DNA polynucleotide" used herein relates to typical forms of DNA polynucleotides, such as genes, exons, open reading frames, etc., because they usually occur in nature, such as in healthy individuals, if the DNA polynucleotide is associated with a disease or most individuals in an individual population. Therefore, "a mutant version of a target DNA polynucleotide is a version in which its molecular structure, such as a sequence, has changed compared to the WT version. For example, in the case where a DNA polynucleotide is associated with a disease, a mutant version of the DNA polynucleotide may be associated with the occurrence of the disease, while a WT version may be associated with a healthy state. Except for the differences, the two molecules are generally identical or at least highly similar. Therefore, in the context of the present invention, both are considered to be target DNA polynucleotides. A mixture of two entities as described above may have any ratio that allows the two entities to be identified.

[0045] In a specific embodiment, after providing a mixture of a DNA polynucleotide comprising at least a WT version and a mutant version of a target DNA polynucleotide, an optional step of end repair and A tail addition of the DNA polynucleotide is performed. This step is intended to convert a DNA polynucleotide having a blunt or protruding 3' or 5' end into a DNA polynucleotide comprising a 3'A protruding end, which is phosphorylated and can be used for subsequent ligation reactions. The execution of this step depends largely on the form and source of the DNA polynucleotide. In certain embodiments, it can also be modified and / or adapted to necessity. For example, if an end repair step is not required or a suitable flat end is already present on the DNA polynucleotide, end repair activity can be omitted. Similarly, in the case where a suitable A protruding end is already present in the DNA polynucleotide, A tail addition activity is not required and can be skipped accordingly. End repair can be performed by any suitable end repair enzyme activity, such as DNA polymerase I (preferably its Klenow fragment), T4 DNA polymerase or T4 polynucleotide kinase. Preferably, end repair is performed with T4 DNA polymerase, T4 PNK and Klenow at 20°C. The A-tailing activity can be performed by any suitable A-tailing enzyme activity (such as Taq DNA polymerase or Klenow fragment). Preferably, the A-tailing is performed with Taq DNA polymerase at 65° C. More details can be obtained from suitable literature sources, such as Nucleic Acids Research, 2010, 38, 13, e137.

[0046] In another preferred embodiment, after the end repair and A-tail addition steps of the DNA polynucleotide, or if the DNA polynucleotide already includes suitable A-overhangs, without the end repair and A-tail addition steps, the DNA polynucleotide having a stem-loop oligonucleotide is subjected to a circularization step.

[0047] Typically, the stem-loop oligonucleotide is first connected to the DNA polynucleotide. Connection preferably occurs at both ends of the DNA polynucleotide. Further preferably, the connection uses a 3' overhang nucleotide at the 3' end of the double-stranded DNA polynucleotide, more preferably at the 3'A overhang of the double-stranded DNA polynucleotide. In a typical embodiment, the stem-loop oligonucleotide includes a 3' overhang, which is compatible with the corresponding overhang on the DNA polynucleotide. In the case of a 3'A overhang at the DNA polynucleotide, the stem-loop oligonucleotide may include a complementary 3'T overhang. The term "connection" as used herein relates to the annealing reaction of the stem-loop oligonucleotide, followed by a suitable bond-forming reaction of the annealed stem-loop oligonucleotide, typically a connection. Connection can be chemical or enzymatic connection. Enzymatic connection is preferred. Chemical connection generally requires the presence of a condensing agent. An example of chemical connection envisioned by the present invention utilizes an electrophilic phosphorothioate thioester group. Further examples include the use of cyanogen bromide as a condensing agent. Enzymatic connection can be performed with any suitable enzymatic ligase known to the technician. Examples of suitable ligases include T4 DNA ligase, E. coli DNA ligase, T3 DNA ligase, and T7 DNA ligase. Alternatively, a ligase such as Taq DNA ligase, Tma DNA ligase, 9°N DNA ligase, T4 polymerase 1, T4 polymerase 2, or a thermostable 5'App DNA / RNA ligase may be used.

[0048] In another step, the DNA polynucleotide can be cyclized after being connected to the stem-loop oligonucleotide, or alternatively in the absence of the connection, that is, the DNA polynucleotide is not connected to the stem-loop oligonucleotide. The term "cyclization" used herein refers to the conversion of a linear nucleic acid molecule to a circular nucleic acid molecule. In principle, cyclization can be achieved by connecting the two ends of the polynucleotide or by melting the polynucleotide while maintaining the continuity of the 3' and 5' ends through the presence of a loop element. Preferably, a strategy based on a circular element is followed. In this embodiment, the cycle includes a melting step, such as raising the reaction temperature to the melting temperature of the DNA polynucleotide. The resulting molecule is a circular ssDNA polynucleotide.

[0049] Particularly preferred is that strand separation and conversion into circular ssDNA molecules is assisted by prior ligation of a DNA polynucleotide and a stem-loop oligonucleotide as defined herein.

[0050] The term "stem-loop oligonucleotide" as used herein refers to a nucleic acid, typically a DNA oligonucleotide, comprising a partially double-stranded segment comprising a double-stranded stem segment and a hairpin or hairpin link segment connecting the double-stranded segments. Therefore, the stem portion typically includes two regions of the same chain that are complementary in nucleotide sequence when read in opposite directions. These segments can base pair and form a double helix ending in an unpaired loop.

[0051] Do not wish to be bound by theory, it is believed that the formation of stem-loop structure depends on the stability of the spiral and loop region of gained.The first prerequisite is usually to have a sequence that can self-fold to form the double helix of pairing.The stability of this spiral depends mainly on its length, the mispairing that it may contain or the quantity of protrusion and the base composition of the pairing region.Because the pairing between guanine and cytosine has three hydrogen bonds, only two hydrogen bonds are compared with adenine-thymine pairing, and they are more stable.In some embodiments, the stem segment comprises more guanine-cytosine pairing than adenine-thymine pairing.

[0052] In addition, the stability of the ring may affect the formation of the stem-loop structure. Preferably, the hairpin loop is not less than three bases, for example 4, 5, 6, 7, 8 or more bases long. It is further preferred that the length of the ring does not exceed about 10 to 12 bases, because large rings are generally unstable. In certain embodiments, the size of the ring can be greater than 12 bases and show other secondary structures, such as pseudoknots. It is particularly preferred that the length of the ring is about 4 to 8 bases. In some embodiments, the ring has a sequence 5'-TNCG-3', that is, it is a four-ring that is stable due to the base stacking interaction of its constituent nucleotides.

[0053] As described above, the stem-loop oligonucleotide according to the present invention may additionally include a barcode sequence or segment in a specific embodiment. As used herein, the term "barcode sequence" or "barcode segment" refers to a sequence that is artificially included in a polynucleotide and is used for identification purposes after a characterization step (e.g., after sequencing). The barcode segment can therefore inform the user which of several samples being characterized, such as sampling. Therefore, the barcode segment includes a unique sequence that is only provided once, i.e., only for one molecule / polynucleotide as described above. The barcode sequence is preferably different from a known natural sequence motif. In other embodiments, it is preferably long enough to avoid confusion with a naturally occurring sequence or a different barcode sequence. According to a preferred embodiment, the length of the barcode sequence is at least 6 to about 12 or more nucleotides. In certain embodiments, the barcode segment may exist once or multiple times in the polynucleotide of the present invention. If there are more than one barcode segment, such as 2, 3, 4, or 5 or more, the distinction (i.e., index order) of each segment is different, thereby allowing two or more independent identification processes. The barcode sequences may for example be advantageously used for multiplexing to different patients or different patient samples etc. Further details will be known to the skilled person or can be obtained from suitable literature sources such as Kozarewa et al., 2011, Methods Mol. Biol. 733, 279-298.

[0054] As mentioned above, in a specific embodiment, the stem-loop oligonucleotide according to the present invention may alternatively or additionally include a restriction enzyme recognition site. The restriction enzyme recognition site may be located at any suitable position in the stem-loop segment. The restriction enzyme recognition site is preferably located in the stem segment of the stem-loop oligonucleotide. It allows the cutting in the oligonucleotide or any molecule connected thereto or including it. For example, after RCA as described herein has been performed, each repeating unit of the amplified DNA polynucleotide includes a unit of at least one restriction enzyme recognition site. It can be subsequently cut (cleave) or cut (cut) at any suitable time point, for example, if the longer concatemer size is reduced to a shorter fragment or a single repeating unit. The term "cutting (" cleaving " or " cleavage ") " used herein refers to double-stranded cutting, i.e., the nick through each chain in a double-stranded nucleic acid molecule, usually performed by a restriction enzyme or restriction endonuclease. The restriction enzyme used for this activity can be any suitable restriction enzyme known to the technician. By cutting at the restriction enzyme recognition site, any suitable end at the cutting site can be produced. Such ends may be sticky ends, i.e. including 5' or 3' overhangs, or may be blunt ends, i.e. without overhangs. Preferably, sticky ends are obtained. Further preferably, the sticky ends are 3' overhangs. In a particularly preferred embodiment, the overhang is a 3' overhang of 1 nucleotide. Even more preferably, the 3' overhang is a 1 nucleotide A overhang. Thus, it is envisaged that a restriction enzyme recognition site is a site that provides a 3' A overhang when cut by a homologous restriction enzyme.

[0055] In a specific group of embodiments, the restriction enzyme recognition site can have the sequence 5'-ACAGT-3' or 5'-TCAGA-3'. According to another embodiment, the restriction enzyme recognition site 5'-ACAGT-3' can be cut at the third position to produce 5'-ACA / GT-3', thereby providing a 3' overhang of 1 nucleotide, more specifically providing a 3'A overhang of 1 nucleotide. Known enzymes Bst4CI, HpyCH4III and Taal can recognize the restriction enzyme recognition site 5'-ACAGT-3'. Therefore, these enzymes can preferably be used in the context of the present invention. According to different embodiments, the restriction enzyme recognition site 5'-TCAGA -3' can be cut at the third position to produce 5'-TCA / GA-3', thereby providing a 3' overhang of 1 nucleotide, more specifically providing a 3'A overhang of 1 nucleotide. Known enzyme Hpyl88I recognizes the restriction enzyme recognition site 5'-TCAGA-3'. Therefore, these enzymes can preferably be used in the context of the present invention.

[0056] In the subsequent steps of the inventive method, DNA polynucleotides are amplified by rolling circle amplification (RCA). The terms "rolling circle amplification" or "RCA" used herein relate to an isothermal enzymatic process, wherein a circular DNA template and a suitable polymerase are generally used to amplify a short DNA polynucleotide to form a long single-stranded DNA polynucleotide. The RCA product is generally a concatemer, which contains several (e.g., 5 to 500) tandem repeats complementary to the circular template. Generally, a suitable DNA polymerase is used for the process. Examples include Phi29 polymerase, Bst polymerase or exo-DNA polymerase. Phi29 polymerase is preferably used. The RCA template used in the context of the present invention is a single-stranded circular DNA molecule. In essence, the reaction is to continuously add nucleotides to a primer annealed to the circular ssRNA template. Therefore, the present invention contemplates that, for example, by using one or more appropriately annealed oligonucleotides, the double-stranded DNA polynucleotide obtained in step (i) is converted into a circular template and the template is converted into a chain form.

[0057] In a preferred embodiment, RCA is performed with one or more oligonucleotides specific to at least a portion of the target DNA polynucleotide. As used herein, the term "specific to the target DNA polynucleotide" relates to sequence complementarity between an oligonucleotide and a DNA polynucleotide, which allows the oligonucleotide to be annealed to the DNA polynucleotide and subsequently subjected to an amplification reaction. Therefore, the term "complementary" or "complementarity" refers to the presence of matching base pairs in opposite nucleic acid chains, i.e., in an oligonucleotide and a DNA polynucleotide. For example, a nucleotide or base T of a complementary or antisense chain binds to a nucleotide or base A in a sense chain, and vice versa; similarly, a nucleotide or base C of a complementary or antisense chain binds to a nucleotide or base G in a sense chain, and vice versa. In certain embodiments of the invention, this completely or perfectly complementary scheme can be modified by the possibility of having single or multiple non-complementary bases or nucleotide extensions in the sense and / or antisense chains. Thus, in order to fall within the concept of a pair of sense and antisense strands, the two strands may be fully complementary or may be only partially complementary, for example showing about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% complementarity between all nucleotides of the two strands or between all nucleotides in a particular segment as defined herein. Non-complementary bases may include one of the nucleotides A, T, G, C, i.e., showing a mismatch, for example, between A and G, or between T and C, or may include any modified nucleobase, including, for example, modified bases as described in WIPO Standard ST.25. Furthermore, the present invention also contemplates complementarity between different nucleic acid molecules, such as complementarity between a DNA strand and an RNA strand, a DNA strand and a PNA strand, a DNA strand and a CNA strand, etc. Preferably, the complementarity between strands or segments as defined herein is full or 100% complementarity.

[0058] "Specific" annealing of oligonucleotides and DNA polynucleotides means that complete or partial complementarity / partial matching is possible, which allows identification of DNA polynucleotides, but in some embodiments, it also accepts the presence of mismatched nucleotides. For example, in the case where annealing occurs in different sections of DNA polynucleotides, it is possible to carry out specific annealing with WT target DNA polynucleotides and mutant target DNA polynucleotides as defined herein. If the mutant is different due to single nucleotide polymorphisms or 2 to 3 nucleotide differences, such annealing is particularly envisioned. In other embodiments, specific annealing can involve complete complementarity, which can be achieved by combining in the sections of DNA polynucleotides, which is not affected by the influence of sequence modifications reflected by the differences of WT and mutant versions of target DNA polynucleotides of the present invention.

[0059] As used herein, in certain embodiments, the term "specific for at least a portion of a target DNA polynucleotide" refers to an oligonucleotide that has at least a complementary overlap with the target DNA polynucleotide. The overlap may be, for example, an overlap of 4, 5, 7, 6, 7, 8, 9, 10, 12, 15, 18 or 20 nucleotides, or any value between the values. The overlap may depend on the size of the oligonucleotide and may be adjusted accordingly. Within the overlap, the match or complementarity between complementary bases is preferably 100%. In an alternative embodiment, the match is less than 100%, such as 99%, 95%, 90%, 85% or less than 85%. The specificity of annealing may be further adjusted by setting the annealing temperature, wherein a higher temperature increases specificity. The hybridization temperature may be calculated by a technician according to known rules depending largely on the sequence involved. It is particularly preferred that the hybridization conditions and oligonucleotide design (including its length) are suitable for the working conditions of the polymerase for RCA as defined herein. For example, in an embodiment using Phi29 polymerase, a processing temperature of about 30°C may be used.

[0060] In certain alternative embodiments, the oligonucleotide may be specific for a stem-loop oligonucleotide sequence as described above, or it may at least partially bind to at least a portion of said stem-loop oligonucleotide sequence.

[0061] In a very specific embodiment, the following steps are performed for RCA: After ligation, an oligonucleotide is added to the ligated DNA. A melting step is then performed as described above. After melting, the temperature is lowered and the oligonucleotide is allowed to bind to its target sequence. A polymerase, such as Phi29 polymerase, is then added to amplify the circular template.

[0062] In some embodiments, only one oligonucleotide that binds to the target DNA polynucleotide can be used for RCA. In other embodiments, more than one oligonucleotide can be used, for example 2, 3, 4, 5, etc. These oligonucleotides may preferably bind at different positions in the target DNA polynucleotide, preferably those that are not affected by sequence modifications reflected by the differences between the WT and mutant versions of the target DNA polynucleotide.

[0063] The oligonucleotide may be of any suitable size, for example 6 to 30 nucleotides. Preferred oligonucleotides are hexamers, heptamers or octamers. If more than one different oligonucleotide is used for RCA, preferably similar or identical sizes are used.

[0064] RCA can be performed at any suitable temperature, for example at room temperature or at a temperature up to 37°C. The temperature may be a constant temperature. RCA can further be performed in any suitable environment, for example in solution or on a solid support. In particular embodiments, RCA reactions in complex biological environments (such as on a cell surface) are also envisioned.

[0065] In another embodiment, RCA can be performed until the amplified DNA polynucleotide has a determined, preferably predetermined size. Size can depend on the subsequent activity planned for the DNA polynucleotide obtained. For example, if the characterization of DNA polynucleotides is to be performed using sequencing techniques based on transmembrane pores, long amplification is preferred. In alternative embodiments, if different NGS methods are to be performed, which generally require short input polynucleotides, short amplicons can be obtained. The size of the amplicon can be at least 300 nucleotides, preferably in the range of about 300 nucleotides to about 10000 nucleotides, more preferably, it can be in the range of about 300 to 7000 nucleotides, such as 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500 or 7000 or any value between the above-mentioned sizes. In a most preferred embodiment, it may be at least about 3000 nucleotides in size. In a further preferred embodiment, short fragments may also be obtained by cleaving longer amplicons, for example via restriction enzyme recognition sites present in the stem-loop structure as described herein.

[0066] The size of the amplification product can be controlled by any suitable means, for example using temperature control, such as a heat denaturation step, or adding inhibitory molecules, adding EDTA, or adding proteases, etc. Preferably, the size of the amplification product is controlled by using a heat denaturation step.

[0067] In certain further embodiments, the RCA contemplated by the present invention may also include a multiplex amplification step, wherein multiple oligonucleotides, e.g., as defined herein, hybridize or anneal to the same target DNA polynucleotide circle, thereby allowing the simultaneous generation of multiple RCA products. Similarly, hyperbranched RCA may be performed, wherein the RCA product is used as a template for further amplification with a second or third set of oligonucleotides.

[0068] Monitoring and detecting the RCA process is also contemplated in the present invention. This can be achieved by incorporating fluorescent dyes into the RCA product, for example by fluorophore-coupled dTNPs or hybridization of complementary strands linked to fluorophores. Detection can thus be performed by means of fluorescence spectroscopy, flow cytometry or microscopy. It is also contemplated to use gold nanoparticles, magnetic beads or quantum dots to detect the RCA product.

[0069] Preferably, the monitoring and detection of the RCA process is performed by means of gel electrophoresis analysis. Further detailed information on RCA can be obtained from appropriate literature sources, such as Ali et al., 2014, Chem. Soc. Rev., 43, 3324-3341.

[0070] In a specific embodiment, the rolling circle amplification product obtained can be repaired using T7 endonuclease and DNA polymerase. For example, T7 endonuclease I and T4 DNA polymerase activity can be used to remove mispairing structures and for repair purposes. Alternatively, ligase activity can also be used for this purpose.

[0071] In another step of the method of the present invention, a synthetic single-stranded guide RNA (sgRNA) specific for the WT version and a sgRNA-guided nucleic acid binding protein are used to recognize and cleave the WT version of the target DNA polynucleotide.

[0072] Typically, this step is based on the use of CRISPR / Cas systems. As used herein, the term "CRISPR / Cas system" relates to a biochemical method for specific cutting and modification of nucleic acids. For example, CRISPR / Cas systems can be used to insert, remove or shut down genes in a genome, or to change nucleotides in a gene or nucleic acid molecule. The concept of the CRISPR / Cas system and the effect of the active step have multiple similarities with RNA interference, because short RNA fragments of about 18 to 20 nucleotides mediate the binding to the target in two bacterial defense mechanisms. In the CRISPR / Cas system, RNA-guided nucleic acid binding proteins, such as Cas proteins, are usually combined with certain RNA sequences as ribonucleoproteins. For example, Cas endonucleases (such as Cas9, Cas5, Csn1 or Csxl2 or derivatives thereof) can be combined to certain RNA sequences repeated by crRNA, and DNA is cut in the immediate vicinity of these sequences. It is not desirable to be bound by theory, and it is believed that crRNA repeats form secondary RNA structures, which are then combined by nucleic acid binding proteins (such as Cas), which change their protein folding, thereby allowing target DNA to be combined by RNA. In addition, a PAM motif, i.e., a protospacer adjacent motif, must be present in the target DNA to activate nucleic acid binding proteins (e.g., Cas). DNA is usually cut by three nucleotides before the PAM motif. The crRNA repeat sequence is usually followed by a sequence that binds to the target DNA, i.e., a crRNA spacer; two sequences, i.e., crRNA repeat motifs and target binding segments, are usually labeled as "crRNA". The second part of crRNA (target binding segment) is a crRNA spacer sequence with a variable transfer function. It is complementary to the target DNA and binds to the target DNA. Additional RNA, tracrRNA, or trans-acting CRISPR RNA is also required. TracrRNA is partially complementary to crRNA so that they bind to each other. TracrRNA is usually combined with precursor crRNA to form an RNA double helix and is converted into an active form by RNase III. These properties allow binding to DNA and cutting by the endonuclease function of nucleic acid binding proteins (e.g., Cas) near the binding site.

[0073] As used herein, the term "synthetic single-stranded guide RNA (sgRNA)" or "single-stranded guide RNA (sgRNA)" refers to an artificial or synthetic combination of crRNA and tracrRNA sequences of the CRISPR / Cas system as described above. Typically, the sgRNA includes a target-specific sequence that can be used to guide the DNA binding protein to the binding site. The target-specific sequence can have any suitable length. Preferably, the length is between about 19 and 30 nucleotides. More preferably, the sequence has a length of 20 nucleotides.

[0074] As described in Jinek et al., 2012, Science, 337, 816-821, crRNA and tracrRNA can be combined into functional varieties (sgRNA) that achieve the above two activities (crRNA and tracrRNA). For example, nucleotides 1-42 of crRNA-sp2, nucleotides 1-36 of crRNA-sp2, or nucleotides 1-32 of crRNA-sp2 can be combined with nucleotides 4-89 of tracrRNA. Other options for obtaining sgRNA can be obtained from Nowak et al., 2016, Nucleic Acids Research, 44, 20, 9555–9564. For example, sgRNAs including different forms of upper stem structures can be provided, or wherein the spacer sequence is differentially truncated from the standard 20 nucleotides to 14 or 15 nucleotides. Further envisioned variants include those that remove the putative RNAP III terminator sequence from the lower stem. A variant is also envisioned in which the upper stem is extended to increase the stability of the sgRNA and enhance its assembly with the nucleic acid binding protein (e.g., Cas protein) guided by the sgRNA. According to other embodiments of the present invention, the sequence and form of the sgRNA may vary according to the form or identity of the nucleic acid binding protein (e.g., Cas protein) guided by the sgRNA. Therefore, depending on the source of the nucleic acid binding protein guided by the sgRNA, different combinations of sequence elements can be used. The present invention further contemplates any future developments in this case, and includes any modification or improvement of the sgRNA-nucleic acid-binding protein interaction that exceeds the information that can be derived from Jinke et al., 2012 or Nowak et al., 2016. In a specific embodiment, the sgRNA to be used may have a sequence of any one of SEQ ID NO: 1 to 3.

[0075] The use of Streptococcus pyogenes sgRNA is particularly preferred, for example, in a commercially available kit, such as the EnGen sgRNA synthesis kit provided by New England Biolabs. Similar sgRNA forms from other commercial suppliers, or separately prepared sgRNAs are also contemplated. If used with Streptococcus pyogenes in the form of a homologous nucleic acid binding protein, such sgRNA can be derived from the sequence of SEQ ID NO: 1. Alternatively, if used with Staphylococcus aureus in the form of a homologous nucleic acid binding protein, the sgRNA can be derived from the sequence of SEQ ID NO: 2. In another alternative, if used with Streptococcus thermophilus in the form of a homologous nucleic acid binding protein, the sgRNA can be derived from the sequence of SEQ ID NO: 3.

[0076] The central principle of the present invention is to use a sequence that binds to a target DNA segment within a sgRNA, wherein the binding sequence is specific for the WT version of the target DNA polynucleotide and is therefore able to recognize the sequence and distinguish it from other sequences, in particular mutant sequences that are different from the binding segment. According to the CRSPR / Cas method as defined above, the WT sequence that has been recognized by the sgRNA can then be cleaved by applying or adding a suitable sgRNA-guided nucleic acid binding protein.

[0077] In a preferred embodiment, the "sgRNA-guided nucleic acid binding protein" used herein is a DNA-binding Cas protein. Examples of such DNA-binding Cas proteins are Cas2, Cas3, Cas5, Csnl or Csxl2 or Cas9. Derivatives or mutants thereof are also contemplated. In a particularly preferred embodiment, the sgRNA-guided nucleic acid binding protein is derived from the Cas9 protein family or a derivative thereof. Even more preferably, the sgRNA-guided nucleic acid binding protein is Cas9 or a derivative thereof. The derivative is preferably a functional derivative having nuclease activity. The present invention further contemplates the use of Cas9 derived from different bacterial sources. For example, the Cas9 protein may be derived from Streptococcus pyogenes, Staphylococcus aureus or Streptococcus thermophilus. Preferably, Cas9 is a Streptococcus pyogenes Cas9 protein. More detailed information on Cas protein forms and uses can be obtained from appropriate literature sources, such as Jiang and Doudna, 2017, Annu. Rev. Biophys., 46, 505-529; Makarova et al., 2011, Biology Direct, 6, 38; or Wang et al., 2016, Annu. Rev. Biochem., 85, 22.1-22.38.

[0078] The activity guided by sgRNA in RCA concatemers usually results in the provision of several small fragments, for example, due to the sequence introduced by the repetitive RCA method, whose length corresponds to the original circular template of RCA. Therefore, a significant size difference between the uncut (mutated) molecule and the cut (WT) molecule is obtained. The term "uncut" molecule or polynucleotide used herein refers to a target DNA polynucleotide that is not recognized by the specific sgRNA defined herein. Such polynucleotides may include any sequence differences from the sgRNA binding segment from the WT sequence. In certain embodiments, the sequence difference is a single nucleotide polymorphism. It is also envisioned that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides are inserted or deleted, etc.

[0079] In the next step of the method, the size difference as above is utilized, wherein according to the size difference between the two DNA polynucleotide forms, the uncut mutation target DNA polynucleotide is separated from the WT fragment of cutting. The size selection step can be performed by any suitable method. For example, a method based on agarose gel or polyacrylamide gel or a method based on beads can be used. Particularly preferably, magnetic beads are used, which can be combined with DNA polynucleotides of different lengths under suitable conditions.

[0080] The obtained target polynucleotide, ie, the DNA polynucleotide comprising the mutated sequence motif, can then be purified, stored and / or used for further activities.

[0081] In the final step of the method, the uncut mutant target DNA polynucleotide is characterized. The term "characterization" as used herein relates to the determination of certain characteristics of the DNA polynucleotide. One of the characteristics to be determined according to the present invention is the length of the DNA polynucleotide. In another embodiment, another characteristic of the DNA polynucleotide to be determined according to the present invention is the GO content of the DNA polynucleotide. It is also envisioned to identify certain motifs or sequence extensions or their absence indicating a specific function, or to identify the identity of the DNA polynucleotide. Particularly preferred is the characterization of the DNA polynucleotide sequence.

[0082] As used herein, the term "characterization of a sequence" refers to any suitable sequencing method known to the skilled person. Preferably, next generation sequencing (NGS) or second generation sequencing techniques can be used, which are generally large-scale parallel sequencing methods performed in a highly parallel manner. For example, sequencing can be performed on platforms such as Roche 454, GS FLX Titanium, Illumina, Life Technologies Ion Proton, Oxford Nanopore Technologies, Solexa, Solid or Helicos Biosciences Heliscope systems according to parallel sequencing methods. In some embodiments, sequencing can also include additional preparation of polynucleotides, sequencing, and subsequent imaging and initial data analysis steps.

[0083] The preparation step for sequencing analysis can for example include cutting the polynucleotide with a restriction enzyme having a homologous restriction enzyme recognition site, preferably in a stem-loop oligonucleotide as described herein. Alternatively, the polynucleotide can be randomly broken into smaller sizes. Thereby generating a sequencing template, such as a fragment template. Therefore, the uncut tandem DNA polynucleotide can be reduced in size to be compatible with a homologous sequencing method. It is also envisioned that the uncut tandem DNA polynucleotide is directly sequenced with a suitable sequencing technique.

[0084] Spatially separated templates can be, for example, attached or fixed on a solid surface, which allows sequencing reactions to be performed simultaneously. In a typical example, a library of nucleic acid fragments is produced, and a transfer sub containing a universal primer site is connected to the end of the fragment. Subsequently, the fragment is denatured into a single strand and captured by beads. After amplification, a large number of templates can be attached or fixed in a polyacrylamide gel, or chemically cross-linked to a glass surface of an amino coating, or deposited on a separate titration plate. Alternatively, solid phase amplification can be used. In this method, forward and reverse primers are usually connected to a solid support. The surface density of the amplified fragment is defined by the ratio of primer to template on the support. This method can produce millions of spatially separated template clusters, which can be hybridized with universal sequencing primers to perform large-scale parallel sequencing reactions. Other suitable selections include a variety of displacement amplification methods. Suitable sequencing methods include, but are not limited to, circular reversible termination (CRT) or by synthetic sequencing (SBS) of Illumina, by connecting sequencing (SBL), single molecule addition (pyrophosphate sequencing) or real-time sequencing. Exemplary platforms using the CRT method are Illumina / Solexa and HelicoScope. Exemplary SBL platforms include Life / APG / SOLiD to support oligonucleotide ligation detection. Exemplary pyrophosphate sequencing platforms are Roche / 454. Exemplary real-time sequencing platforms include Pacific Biosciences platforms and Life / Visi-Gen platforms. Other sequencing methods for obtaining a large amount of parallel nucleic acid sequence data include nanopore sequencing, hybrid sequencing, sequencing based on nano transistor arrays, sequencing based on scanning tunneling microscopes (STM), or sequencing based on nanowire molecular sensors. More details about sequencing methods will be known to technicians, or can be obtained from suitable literature sources, such as Goodwin et al., 2016, Nature Reviews Genetics, 17, 333-351; van Dijk et al., 2014, Trends in Genetics, 9, 418-426; or Feng et al., 2015, Genomics Proteomics Bioinformatics, 13, 4-16.

[0085] The size reduction of uncut DNA polynucleotide can be obtained by shearing or fragmentation method according to any suitable scheme known to the technician. Such methods include restriction enzyme digestion, adaptive focused acoustic wave shearing (AFA) or Covaris shearing, use atomization force, ultrasonic treatment, point sheet (point-sink) shearing or use French press (French press) shearing program. As mentioned above, preferably restriction enzyme digestion is utilized in stem-loop oligonucleotide. The size of the polynucleotide obtained is further preferably similar or within a predetermined range. The scope of envision is about 120 to about 400 nucleotides. Particularly preferred is the size of about 150 to 300 nucleotides.

[0086] In a particularly preferred embodiment, the characterization step (v) as described above includes additional sub-steps related to the characterization of the transmembrane pore base sequence. Typically, such characterization includes the following steps: (va) connecting a transfer polynucleotide associated with a DNA translocation enzyme and at least one cholesterol tether segment to a mutant target DNA polynucleotide obtained in step (iv); (vb) contacting the modified DNA polynucleotide obtained in step (va) with a transmembrane pore so that the DNA translocation enzyme controls the DNA polynucleotide to move through the transmembrane pore, and the cholesterol tether anchors the DNA polynucleotide near the transmembrane pore; and (vc) performing one or more measurements during the movement of the DNA polynucleotide through the transmembrane pore, wherein the measurement indicates one or more characteristics of the DNA polynucleotide, thereby characterizing the target DNA polynucleotide.

[0087] The term "transfer polynucleotide complex" as used herein refers to a complex of polynucleotides, which in particular includes a sequence that facilitates the entry of a DNA translocase into a transmembrane pore. In a specific embodiment of the invention, the transfer polynucleotide complex includes a pair of two at least partially complementary polynucleotides. Particularly preferably, the transfer polynucleotide complex is attached to two strands of a DNA polynucleotide to characterize the two strands.

[0088] In certain embodiments, the portion of the transfer subcomplex associated with the DNA translocation enzyme may include a leader sequence. Typically, as described herein, the leader sequence penetrates the transmembrane pore. The leader sequence may further include other fragments, such as one or more spacers. The spacer may, for example, include a sequence that can pause the DNA translocation enzyme. Particularly preferably, the leader sequence includes a binding site for the DNA translocation enzyme. As used herein, the term "DNA translocation enzyme binding site" includes a certain length of DNA or DNA analog sequence that allows one or more DNA translocation enzymes to bind thereto. The length of the binding site generally depends on the number of DNA translocation enzymes to be bound thereto. The region that the DNA translocation enzyme can bind to is preferably a polynucleotide, such as DNA, a modified polynucleotide (e.g., abasic DNA), PNA, LNA, or polyethylene glycol (PEG). Preferably, the binding site for the DNA translocation enzyme is a single-stranded non-hybridized region. Therefore, in a preferred embodiment, the transfer sub-polynucleotide complex is pre-bound to one or more DNA translocation enzymes. As used herein, the term "DNA translocation enzyme" relates to a motor protein that is capable of interacting with a transmembrane pore as described herein and thus transporting a polynucleotide as a single-stranded entity through the pore, i.e., controlling the translocation of a polynucleotide as described herein, e.g., a DNA polynucleotide as defined above, preferably a tandem DNA polynucleotide obtained according to the invention. Examples of suitable translocation enzymes include DNA helicases, such as Hel308 helicase, RecD helicase, XPD helicase or Dda helicase.

[0089] In further embodiments, the leader sequence may include one or more blocking sites that can prevent backward movement of the DNA translocase or prevent the enzyme from sliding out of the transmembrane pore.

[0090] The transfer sub-polynucleotide complex can be further associated with or include a tether segment. As used herein, the term "tether segment" relates to an element that can couple the transfer sub-polynucleotide complex and any other element connected thereto to a double-layer membrane. Coupling is typically instantaneous, and is transmitted by any suitable molecule, preferably a cholesterol entity or a fatty acid, more preferably a cholesterol entity, such as a cholesterol-TEG molecule. Therefore, coupling helps to anchor the transfer sub-polynucleotide complex and its associated elements at or near the transmembrane pore, thereby allowing the DNA polynucleotide introduced in the opposite position to enter the transmembrane pore and characterize. Particularly preferably, the tether segment is provided on both chains of the DNA polynucleotide to allow both chains to be characterized.

[0091] Alternative compounds that can be used to couple to the membrane include biotin, thiol or lipid. In addition to the coupling functionality, the tether generally includes a non-RNA polynucleotide that is connected to the coupling entity, such as a cholesterol entity. The tether segment may further include one or more joint segments, such as a portion of variable length, which may be used to increase the distance between the target DNA polynucleotide and the transmembrane pore to facilitate its characterization. In other embodiments, the joint may include a DNA translocation enzyme binding site as defined herein above.

[0092] The connection of the polynucleotide complex to the polynucleotide obtained in the previous step can be carried out by a connection step. Alternatively, any other suitable connection method can be used, such as chemical connection by click chemistry or covalent bonding, etc. Preferably, the connection is carried out so that the DNA translocation enzyme is connected to the DNA polynucleotide to be characterized, and the tethering element is connected to the complementary strand.

[0093] In a further step, the modified DNA polynucleotide obtained in the previous step (va) is contacted with the transmembrane pore so that the DNA translocation enzyme controls the DNA polynucleotide to move through the transmembrane pore, and the cholesterol tether anchors the DNA polynucleotide near the transmembrane pore. Generally, the function of the tether anchoring described herein is to bring the molecule to the membrane surface where the transmembrane pore is located. In this case, since the transmembrane pore can be reached more easily, the characterization of the DNA polynucleotide can be promoted. As used herein, the term "transmembrane pore" refers to a protein across a bilayer membrane, which includes an opening that can guide the passage of the polynucleotide. The transmembrane pore can be any suitable protein. Examples of preferred transmembrane proteins include protein pores derived from the following: hemolysin, leukocidin, MspA, MspB, MspC, MspD, CsgG, lysenin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) or WZA. Commercially available transmembrane porins, such as those provided or described by Oxford Nanopore Technology, are also contemplated.

[0094] In the final step (vc), one or more measurements are performed during the DNA polynucleotide moves through the transmembrane pore. The measurement can indicate one or more features of the DNA polynucleotide, which allows characterization of the target DNA polynucleotide as defined above, particularly the sequence of the DNA polynucleotide. As used herein, the term "measurement" relates to optical and / or electrical measurements, preferably to electrical measurements at the transmembrane pore. Typically, the electric current passing through the transmembrane pore is measured when the target DNA polynucleotide passes through the transmembrane pore. The measured electric current typically indicates one or more features of the polynucleotide analyzed. For example, the method can be performed using the equipment described in the prior art, such as the equipment disclosed in principle in WO2008 / 102120 or its derivative or modified version. Typically, the method can be performed using a patch clamp or voltage clamp to detect the change in electric current on the transmembrane pore when the polynucleotide moves through the hole. In certain embodiments, the measurement includes the use of charge carriers, such as metal salts, chloride salts, ionic liquids, organic salts, in particular NaCl, KCl, CsCl; further contemplated use of suitable buffers, such as HEPES, Tris-HCl, etc.; further contemplated use of nucleotides, such as AMP, ADP, ATP, dAMP, dADP, dATP, etc., which can be used for translocase activity; and enzyme cofactors, such as Mg 2+ , Ca 2+ 、Co 2+ Divalent metal ions.

[0095] On the other hand, the present invention relates to a kit comprising one or more oligonucleotides specific to at least a portion of a target DNA polynucleotide, preferably as defined herein above, a synthetic single-stranded guide RNA (sgRNA) specific to a WT version of a target DNA polynucleotide, preferably as defined above, and a nucleic acid binding protein guided by sgRNA, preferably as defined above. The kit is preferably used to characterize a target DNA polynucleotide. The features of the method as defined above are also applicable to the kit of the present invention. The kit may, for example, include reagents and components as defined in one or more steps of the method of the present invention or known to those skilled in the art. For example, the kit may include reagents or components for performing RCA based on one or more oligonucleotides defined herein. In addition, it may include reagents and components for subsequent repair of the RCA product, such as T7 endonucleases and / or DNA polymerases as described herein, and optional ligases. The kit may alternatively or additionally include reagents and components for cutting a WT version of a target DNA polynucleotide with a nucleic acid binding protein guided by sgRNA as defined herein. In different embodiments, the kit may include or may additionally include reagents or components for size selection. The kit may typically include a suitable buffer solution, a marker or a washing solution, etc. Furthermore, the kit may include a certain amount of known nucleic acid molecules or proteins, which may be used for calibration of the kit or as an internal control. The corresponding components are known to the skilled person.

[0096] In another preferred embodiment, the kit may include or additionally include components necessary for sequencing reactions. It is particularly preferred to provide components and reagents required for transmembrane pore sequencing methods in the kit. For example, the kit may include or additionally include reagents or components for connecting the transfer sub-polynucleotide complex associated with DNA translocation enzyme and at least one cholesterol tether segment to polynucleotides as described herein. In another embodiment, the kit may include or additionally include reagents or components for contacting the target DNA polynucleotide as defined herein with the transmembrane pore, so that the DNA translocation enzyme controls the target DNA polynucleotide to move through the transmembrane pore and the cholesterol tether anchors the target DNA polynucleotide near the transmembrane pore. In another embodiment, the kit may include or additionally include reagents or components for measuring one or more times during the target DNA polynucleotide moves through the transmembrane pore, wherein the measurement indicates one or more features of the target DNA polynucleotide, as described above, so as to characterize the target DNA polynucleotide. The kit may further include two or more components or reagent groups as defined above, such as components or reagents for performing 2 steps as defined herein, 3 steps as defined herein, 4 steps as defined herein, etc.

[0097] Additionally, the kit may include an instruction leaflet and / or may provide information regarding its usage and the like.

[0098] It is also contemplated that a device for performing the above-mentioned method steps. The device can, for example, be composed of different modules that can perform one or more steps of the method of the present invention. These modules can be combined in any suitable manner, for example, they can be present in a single location or separated. It is also contemplated that the method is performed at different time points and / or at different locations. Some steps of the method as defined herein can be interrupted or paused afterwards, wherein reagents or products, etc. are suitably stored in, for example, a refrigerator or a cooling device. If these steps are performed in a specific module of the device as defined herein, the module can be used as a storage tool. The module can further be used to transport the reaction product or reagent to different locations, such as different laboratories, etc.

[0099] The present invention also contemplates the use of one or more of the kit components as described above in characterizing a target DNA polynucleotide.

[0100] Now turn to Figure 1 , a schematic diagram of the steps of characterizing a target DNA polynucleotide using rolling circle amplification (RCA) and a synthetic single-stranded guide RNA (sgRNA) according to one embodiment of the present invention is shown. In the first step, a DNA polynucleotide 1 representing a mixture of target DNA polynucleotides is provided. The DNA polynucleotide 2 is modified by end repair and T tail addition 3 activities. Subsequently, a stem-loop oligonucleotide 4 with a compatible 3'T overhang is connected to the DNA polynucleotide 5. This step produces a ds DNA polynucleotide 6 having both ends including a stem-loop oligonucleotide 4. Subsequently, an oligonucleotide 8 specific to at least a portion of the target DNA polynucleotide is annealed 7 to the modified DNA polynucleotide 6. The next step is rolling circle amplification (RCA) 9, and then a ds DNA polynucleotide 10 is provided by the activity of a DNA polymerase and an optional ligase. This step produces a mixture of tandem DNA polynucleotides representing a WT sequence 12 or a mutant sequence 13. In the case where the DNA polynucleotide represents a WT sequence 12, the concatemer is processed 14 into smaller fragments 15 with sgRNA and a nucleic acid binding protein (such as Cas 9) guided by the sgRNA. In case the DNA polynucleotides represent mutant sequences, they remain uncleaved 13. The uncleaved mutant DNA polynucleotides are then separated from the WT sequence by size selection 16. They can be further modified and used in a transmembrane pore 17 based sequencing method 18, which is performed in a suitable sequencer 19.

[0101] Reference numerals

[0102] 1 DNA polynucleotide representing the target DNA polynucleotide mixture

[0103] 2 Modification of DNA polynucleotides

[0104] 3 DNA polynucleotide added to tail A

[0105] 4 stem ring oligonucleotides

[0106] 5. Linking of stem-loop oligonucleotides to DNA polynucleotides

[0107] 6 ds DNA polynucleotides with stem-loop oligonucleotides at both ends

[0108] 7 Annealing reaction

[0109] 8. An oligonucleotide specific for at least a portion of a target DNA polynucleotide

[0110] 9 Rolling Ring Amplification (RCA)

[0111] 10 Provide ds DNA polynucleotides

[0112] 11 Mixture of tandem DNA polynucleotides

[0113] 12 Tandem body of WT sequence

[0114] 13 Tandems of mutation sequences

[0115] 14. Processing with sgRNA and sgRNA-guided nucleic acid binding proteins

[0116] 15 Tandem Fragment of WT Sequence

[0117] 16 Selection of size of mutation tandem

[0118] 17 Transmembrane pores

[0119] 18 Sequencing Reactions

[0120] 19 Sequencing Machine

[0121] The following figure is for illustration only. Therefore, it should be understood that the figure should not be interpreted as limiting. Those skilled in the art will clearly be able to conceive further modifications to the principles set forth herein.

Claims

1. A method for characterizing a mutant target DNA polynucleotide, comprising: (i) providing a mixture of DNA polynucleotides, wherein the mixture of DNA polynucleotides comprises at least a wild-type (WT) version and a mutant version of the DNA polynucleotide; (ii) amplifying the mixture of DNA polynucleotides of step (i) by rolling circle amplification (RCA) to provide a library of amplified and concatenated DNA polynucleotides; (iii) recognizing and cleaving the WT version of the target DNA polynucleotide by using a synthetic single-stranded guide RNA (sgRNA) specific for the WT version and a sgRNA-guided nucleic acid binding protein; (iv) size selecting the uncleaved mutant target DNA polynucleotide; and (v) characterizing the uncleaved mutant target DNA polynucleotide, wherein: After step (i), the method further comprises a step (ia) of repairing the ends of the DNA polynucleotide and adding an A tail; After step (ia), the method further comprises the step (ib) of circularizing the DNA polynucleotide using a stem-loop oligonucleotide, wherein the stem-loop oligonucleotide comprises a barcode sequence and a restriction enzyme recognition site; and The method is for non-diagnostic purposes.

2. The method according to claim 1, wherein: The nucleic acid binding protein guided by the sgRNA is Cas9.

3. The method according to claim 1, wherein: Step (v) includes the following sub-steps: (va) ligating an adapter polynucleotide associated with a DNA translocase and at least one cholesterol tether segment to the mutant target DNA polynucleotide obtained in step (iv); (vb) contacting the modified DNA polynucleotide obtained in step (va) with a transmembrane pore, so that the DNA translocation enzyme controls the movement of the DNA polynucleotide through the transmembrane pore, and the cholesterol tether anchors the DNA polynucleotide near the transmembrane pore; and (vc) taking one or more measurements during movement of the DNA polynucleotide through the transmembrane pore, wherein the measurements are indicative of one or more characteristics of the DNA polynucleotide, thereby characterizing the target DNA polynucleotide.

4. The method according to claim 1, wherein: The rolling circle amplification is performed using one or more oligonucleotides specific for at least a portion of the target DNA polynucleotide.

5. The method of claim 4, wherein the one or more oligonucleotides specific for at least a portion of the target DNA polynucleotide are hexamers, heptamers and / or octamers. The method of claim 1 , wherein the rolling circle amplification is performed until the amplified DNA polynucleotide has a size of at least 300 nucleotides.

7. The method of claim 1, wherein the rolling circle amplification is performed until the amplified DNA polynucleotide has a size of at least 3000 nucleotides.

8. The method according to claim 4, wherein the rolling circle amplification product is obtained by repairing using T7 endonuclease and DNA polymerase.

9. The method according to claim 4, wherein the rolling circle amplification product is obtained by repairing using T7 endonuclease, DNA polymerase and ligase.

10. The method according to any one of claims 1 to 9, wherein: The target DNA polynucleotide represents a gene, one or more exons of a gene, an intergenic region, a non-transcribed regulatory region and / or an open reading frame or a sub-portion thereof; or a group of different genes, one or more exons of a group of different genes, a group of intergenic regions, a group of non-transcribed regulatory regions and / or a group of open reading frames or a sub-portion thereof, or any combination of any of the aforementioned elements.

11. The method according to any one of claims 1 to 9, wherein the target DNA polynucleotide is cell-free DNA (cfDNA).

12. The method of claim 11, wherein the target DNA polynucleotide is derived from a liquid biopsy.

13. The method according to any one of claims 1 to 9, wherein the characterization of the DNA polynucleotide is (i) determining the length of the DNA polynucleotide, (ii) determining the identity of the DNA polynucleotide, or (iii) determining the sequence of the DNA polynucleotide.

14. The method according to any one of claims 1 to 9, wherein the characterization of the DNA polynucleotide is determining the sequence of the DNA polynucleotide.

15. The method of claim 3, wherein the DNA translocase is a DNA helicase.

16. The method of claim 15, wherein the DNA helicase is Hel308 helicase, RecD helicase, XPD helicase or Dda helicase.

17. The method of claim 3, wherein the transmembrane pore is a protein pore derived from hemolysin, leukocidin, MspA, MspB, MspC, MspD, CsgG, lysenin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP), or WZA.

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