An inventive method for amplifying at least one RNA which is contained in a sample includes reverse transcribing the at least one RNA using a first primer, adding a dideoxy nucleotide which is modified in the 3′-position with a first partner of a pair of azide and alkyne molecules by action of a template independent polymerase to attach a single 3′-azide- or 3′-alkyne-modified dideoxy nucleotide at the 3′-end of the obtained cDNA, adding an adapter molecule which comprises a polynucleotide sequence and at its 5′-end a second partner of such pair of azide and alkyne molecules and ligating the adapter to the 3′-modified cDNA under formation of a triazole linkage, adding a second primer which is complementary to at least a part of the adapter molecule and which contains at its 3′-end a nucleotide which is complementary to the dideoxy nucleotide at the 3′-end of the cDNA to effect hybridization and binding of the second primer overlapping the triazole linkage, adding a third primer and amplifying the full length cDNA. Variations of this method are also disclosed. Uses of such method especially for preparing a full length RNA library and for sequencing of a plurality of RNAs contained in a sample, as well as reagent kits for performing such methods are also disclosed and included in the invention.

An inventive method for amplifying at least one RNA which is contained in a sample includes reverse transcribing the at least one RNA using a first primer, adding a dideoxy nucleotide which is modified in the 3′-position with a first partner of a pair of azide and alkyne molecules by action of a template independent polymerase to attach a single 3′-azide- or 3′-alkyne-modified dideoxy nucleotide at the 3′-end of the obtained cDNA, adding an adapter molecule which comprises a polynucleotide sequence and at its 5′-end a second partner of such pair of azide and alkyne molecules and ligating the adapter to the 3′-modified cDNA under formation of a triazole linkage, adding a second primer which is complementary to at least a part of the adapter molecule and which contains at its 3′-end a nucleotide which is complementary to the dideoxy nucleotide at the 3′-end of the cDNA to effect hybridization and binding of the second primer overlapping the triazole linkage, adding a third primer and amplifying the full length cDNA. Variations of this method are also disclosed. Uses of such method especially for preparing a full length RNA library and for sequencing of a plurality of RNAs contained in a sample, as well as reagent kits for performing such methods are also disclosed and included in the invention.

Provided is a method including extending a ssDNA by sequentially adding a plurality of modified nucleoside triphosphates to the ssDNA, wherein the base of the modified nucleoside triphosphates includes a primary modification selected from (i) a primary polynucleotide attached to the base of the modified nucleoside triphosphate, and (ii) a site on the base for covalent attachment of a primary polynucleotide to the base, further comprising covalently attaching a primary polynucleotide to the base after the polymerizing.

Provided is a method including extending a ssDNA by sequentially adding a plurality of modified nucleoside triphosphates to the ssDNA, wherein the base of the modified nucleoside triphosphates includes a primary modification selected from (i) a primary polynucleotide attached to the base of the modified nucleoside triphosphate, and (ii) a site on the base for covalent attachment of a primary polynucleotide to the base, further comprising covalently attaching a primary polynucleotide to the base after the polymerizing.

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template.

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template.

The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides employing photocleavable linkages. In some embodiments, such methods include using 3′-O—NH2-dNTP monomers which may react with photocleavage products having free ketone to allow synthesis and purification on the same or an added support.

The present invention is directed to methods and kits for template-free enzymatic synthesis of polynucleotides employing photocleavable linkages. In some embodiments, such methods include using 3′-O—NH2-dNTP monomers which may react with photocleavage products having free ketone to allow synthesis and purification on the same or an added support.

The invention relates to a nucleic acid detection system, a diagnostic device, use of the nucleic acid detection system as a diagnostic agent, a kit-of-parts for detecting nucleic acids, a method for detecting nucleic acids, and a method for diagnosing a disease state of a subject. The nucleic acid detection system comprises a CRISPR-Cas system which comprises an effector protein and one or more guide RNAs having a guide sequence, the guide sequence being capable of targeting the effector protein to a target sequence of a target, and the effector protein exhibiting target-activated nucleic acid cleavage activity capable of cleaving nucleic acid reporter molecules to generate nucleic acid fragments; and a polymerase exhibiting catalytic activity capable of transferring nucleotides to the fragments to form polynucleotide tails, wherein preferably the detection system is a nucleic acid fluorescence detection system.

The invention relates to a nucleic acid detection system, a diagnostic device, use of the nucleic acid detection system as a diagnostic agent, a kit-of-parts for detecting nucleic acids, a method for detecting nucleic acids, and a method for diagnosing a disease state of a subject. The nucleic acid detection system comprises a CRISPR-Cas system which comprises an effector protein and one or more guide RNAs having a guide sequence, the guide sequence being capable of targeting the effector protein to a target sequence of a target, and the effector protein exhibiting target-activated nucleic acid cleavage activity capable of cleaving nucleic acid reporter molecules to generate nucleic acid fragments; and a polymerase exhibiting catalytic activity capable of transferring nucleotides to the fragments to form polynucleotide tails, wherein preferably the detection system is a nucleic acid fluorescence detection system.