The invention relates to methods for template-independent synthesis of nucleic acids, comprising iteratively contacting an initiator sequence comprising a 3′-end nucleotide with a free 3′-hydroxyl group, with at least one nucleoside triphosphate, or a combination of nucleoside triphosphates, in the presence of an archaeal DNA primase or a functionally active fragment and/or variant thereof, thereby covalently binding said nucleoside triphosphate to the free 3-hydroxyl group of the 3-end nucleotide. It also relates to isolated functionally active fragments of archaeal DNA primases which are capable of template-independent terminal nucleotidyl transferase activity but are devoid of a template-independent primase activity.

The invention relates to methods for template-independent synthesis of nucleic acids, comprising iteratively contacting an initiator sequence comprising a 3′-end nucleotide with a free 3′-hydroxyl group, with at least one nucleoside triphosphate, or a combination of nucleoside triphosphates, in the presence of an archaeal DNA primase or a functionally active fragment and/or variant thereof, thereby covalently binding said nucleoside triphosphate to the free 3-hydroxyl group of the 3-end nucleotide. It also relates to isolated functionally active fragments of archaeal DNA primases which are capable of template-independent terminal nucleotidyl transferase activity but are devoid of a template-independent primase activity.

The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.

A method of labeling, and optionally enriching, for a population of target RNA molecules in a mixture of RNAs is provided. In some embodiments, the method may comprise (a) adding a label to the 5′ end of 5′-diphosphorylated or 5′-triphosphorylated target RNA molecules in a sample by incubating the sample with labeled GTP and a capping enzyme; and (b) optionally enriching for target RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag. The label may be an oligonucleotide, which may further comprise an affinity group attached either internally or at 5′ or 3′ end of the oligonucleotide where the oligonucleotide label may be added directly, or indirectly via a reaction with a reactive group to the target RNA.

A method of labeling, and optionally enriching, for a population of target RNA molecules in a mixture of RNAs is provided. In some embodiments, the method may comprise (a) adding a label to the 5′ end of 5′-diphosphorylated or 5′-triphosphorylated target RNA molecules in a sample by incubating the sample with labeled GTP and a capping enzyme; and (b) optionally enriching for target RNA comprising the affinity tag-labeled GMP using an affinity matrix that binds to the affinity tag. The label may be an oligonucleotide, which may further comprise an affinity group attached either internally or at 5′ or 3′ end of the oligonucleotide where the oligonucleotide label may be added directly, or indirectly via a reaction with a reactive group to the target RNA.

The invention relates to a method for modifying a template double stranded polynucleotide, especially for characterisation using nanopore sequencing. The method produces from the template a plurality of modified double stranded polynucleotides. These modified polynucleotides can then be characterised.

The invention relates to a method for modifying a template double stranded polynucleotide, especially for characterisation using nanopore sequencing. The method produces from the template a plurality of modified double stranded polynucleotides. These modified polynucleotides can then be characterised.

Provided are a method for breaking a nucleic acid and adding an adaptor by means of a transposase, and a reagent. The method comprises the following steps: conducting random breaking of a nucleic acid by using a transposase-embedded complex, wherein the transposase-embedded complex comprises a transposase and a first adaptor comprising a transposase identification sequence, and two ends of the broken nucleic acid are separately connected to the first adaptor and are separately provided with a gap; by means of purification or chemical reagent treatment, eliminating the influence of the transposase in the system on a follow-up reaction; connecting to a second adaptor at the gap by using a ligase, wherein a sequence of the second adaptor is different from a sequence of the first adaptor; and conducting a PCR reaction by using primers targeted to and combined with the first adaptor and the second adaptor respectively, so as to obtain a product whose both ends are respectively connected to different adaptor sequences.

Provided are a method for breaking a nucleic acid and adding an adaptor by means of a transposase, and a reagent. The method comprises the following steps: conducting random breaking of a nucleic acid by using a transposase-embedded complex, wherein the transposase-embedded complex comprises a transposase and a first adaptor comprising a transposase identification sequence, and two ends of the broken nucleic acid are separately connected to the first adaptor and are separately provided with a gap; by means of purification or chemical reagent treatment, eliminating the influence of the transposase in the system on a follow-up reaction; connecting to a second adaptor at the gap by using a ligase, wherein a sequence of the second adaptor is different from a sequence of the first adaptor; and conducting a PCR reaction by using primers targeted to and combined with the first adaptor and the second adaptor respectively, so as to obtain a product whose both ends are respectively connected to different adaptor sequences.

The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.