Linear double stranded dna coupled to a single support or a tag and methods for producing said linear double stranded dna
AssigneeCureVac AG
InventorsBenyamin YAZDAN PANAH, Tilmann ROOS, Veronika WAGNER, Carola PONGRATZ
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Abstract
The present invention is concerned with linear double stranded DNA, which is coupled to a single support or a tag at the 3′ end of its non-coding strand and methods for producing said linear double stranded DNA. The present invention further relates to the use of said linear double stranded DNA in an RNA in vitro transcription reaction and also to a method for producing RNA in vitro. The present invention also relates to a bioreactor for RNA in vitro transcription.
Claims21
A linear double stranded DNA comprising a coding strand and a non-coding strand, wherein said DNA comprises (i) a coding sequence element encoded by the coding strand in the direction of from 5′ to 3′ of the coding strand and (ii) an RNA polymerase promotor sequence element upstream of the coding sequence element, wherein said non-coding strand is coupled at its 3′ end to a support or a tag, and wherein said support or tag is the only support or tag coupled to said DNA.
A method for producing linear double stranded DNA comprising a coding strand and a non-coding strand, wherein said non-coding strand is coupled at its 3′ end to a support or a tag, comprising the steps of:
- (a) providing linear double stranded DNA comprising a coding sequence element encoded by the coding strand, followed at the 3′ end by a restriction site element;
- (b) incubating said DNA with (i) a modified deoxynucleotide and (ii) an enzyme capable of adding said modified deoxynucleotide at a 3′ end of a strand in order to provide linear double stranded DNA with a modified deoxynucleotide at the 3′ end of each strand;
- (c) incubating the DNA obtained in step (b) with a restriction endonuclease recognizing said restriction site element in order to obtain linear double stranded DNA with a modified deoxynucleotide at the 3′ end of the non-coding strand;
- (d) coupling the DNA obtained in step (c) via its modified deoxynucleotide to a support or a tag in order to provide linear double stranded DNA, wherein the non-coding strand of said DNA is coupled at its 3′ end to a support or a tag.
A method for producing linear double stranded DNA comprising a coding strand and a non-coding strand, wherein said non-coding strand is coupled at its 3′ end to a support or a tag, comprising the steps of:
- (a) providing linear double stranded DNA comprising a coding sequence element encoded by the coding strand, followed at the 3′ end by a restriction site element;
- (b) incubating said DNA with (i) a modified deoxynucleotide and (ii) an enzyme capable of adding said modified deoxynucleotide at a 3′ end of a strand in order to provide linear double stranded DNA with a modified deoxynucleotide at the 3′ end of each strand;
- (c) coupling the DNA obtained in step (b) via the modified deoxynucleotide at the 3′ end of each strand to a support or a tag;
- (d) incubating the DNA obtained in step (c) with a restriction endonuclease recognizing said restriction element in order to provide linear double stranded DNA, wherein the non-coding strand of said DNA is coupled at its 3′ end to a support or a tag.
10-39. (canceled)
49-62. (canceled)
A kit comprising
- (a) a tag-linked deoxynucleotide;
- (b) a Thermus aquaticus DNA polymerase capable of adding said tag-linked deoxynucleotide to the 3′ end of a strand at a blunt DNA end;
- (c) a counterpart of said tag associating in a highly specific manner with said tag.
Dependencies are inferred from the claim text.