Provided herein is a method of moving a double-stranded polynucleotide with respect to a nanopore using a motor protein. The method allows a portion of the polynucleotide to be interrogated by the pore multiple times. Also provided are polynucleotide adapters and kits comprising such adapters. The methods find use in characterising polynucleotides, for example in sequencing.

Provided herein is a method of moving a double-stranded polynucleotide with respect to a nanopore using a motor protein. The method allows a portion of the polynucleotide to be interrogated by the pore multiple times. Also provided are polynucleotide adapters and kits comprising such adapters. The methods find use in characterising polynucleotides, for example in sequencing.

Provided herein is a method of characterising a target polynucleotide as it moves with respect to a nanopore using a motor protein. Also provided are polynucleotide adapters and kits comprising such adapters. The methods, kits and adapters find use in characterising polynucleotides, for example in sequencing.

Provided herein is a method of characterising a target polynucleotide as it moves with respect to a nanopore using a motor protein. Also provided are polynucleotide adapters and kits comprising such adapters. The methods, kits and adapters find use in characterising polynucleotides, for example in sequencing.

Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule in vitro or in vivo using an RNA-guided DNA endonuclease comprising RNA sequences and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro and using a cassette containing a single repeat-spacer-repeat unit.

Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule in vitro or in vivo using an RNA-guided DNA endonuclease comprising RNA sequences and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro and using a cassette containing a single repeat-spacer-repeat unit.

Provided herein are methods and devices for characterizing a biomolecule parameter by a nanopore-containing membrane, and also methods for making devices that can be used in the methods and devices provided herein. The nanopore membrane is a multilayer stack of conducting layers and dielectric layers, wherein an embedded conducting layer or conducting layer gates provides well-controlled and measurable electric fields in and around the nanopore through which the biomolecule translocates. In an aspect, the conducting layer is graphene.

Provided herein are methods and devices for characterizing a biomolecule parameter by a nanopore-containing membrane, and also methods for making devices that can be used in the methods and devices provided herein. The nanopore membrane is a multilayer stack of conducting layers and dielectric layers, wherein an embedded conducting layer or conducting layer gates provides well-controlled and measurable electric fields in and around the nanopore through which the biomolecule translocates. In an aspect, the conducting layer is graphene.

In an aspect, there is provided a method of detecting the presence of ctDNA from cancer cells in a subject comprising: (a) providing a sample of cell-free DNA from a subject; (b) subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA; (c) optionally adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated, then further optionally denaturing the sample; (d) capturing cell-free methylated DNA using a binder selective for methylated polynucleotides; (e) sequencing the captured cell-free methylated DNA; (f) comparing the sequences of the captured cell-free methylated DNA to control cell-free methylated DNAs sequences from healthy and cancerous individuals; (g) identifying the presence of DNA from cancer cells if there is a statistically significant similarity between one or more sequences of the captured cell-free methylated DNA and cell-free methylated DNAs sequences from cancerous individuals; wherein in at least one of the capturing step, the comparing step or the identifying step, the subject cell-free methylated DNA is limited to a sub-population according to a fragment length metric.

In an aspect, there is provided a method of detecting the presence of ctDNA from cancer cells in a subject comprising: (a) providing a sample of cell-free DNA from a subject; (b) subjecting the sample to library preparation to permit subsequent sequencing of the cell-free methylated DNA; (c) optionally adding a first amount of filler DNA to the sample, wherein at least a portion of the filler DNA is methylated, then further optionally denaturing the sample; (d) capturing cell-free methylated DNA using a binder selective for methylated polynucleotides; (e) sequencing the captured cell-free methylated DNA; (f) comparing the sequences of the captured cell-free methylated DNA to control cell-free methylated DNAs sequences from healthy and cancerous individuals; (g) identifying the presence of DNA from cancer cells if there is a statistically significant similarity between one or more sequences of the captured cell-free methylated DNA and cell-free methylated DNAs sequences from cancerous individuals; wherein in at least one of the capturing step, the comparing step or the identifying step, the subject cell-free methylated DNA is limited to a sub-population according to a fragment length metric.