Method for the 5'-cap-dependent amplification of cDNAs
AssigneeRoche Diagnostics GmbH
InventorsManfred W. Mueller, Wolfgang M. Schmidt
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Abstract
The invention concerns a method for the modification, cloning and amplification of cDNAs which are complete at their 5′ end which is essentially characterized in that the first strand cDNA synthesis is carried out in the presence of manganese2+ ions or manganese2+ is added as an additive at a later time. The CAP structure at the 5′ end of the reversely transcribed mRNA triggers the attachment of deoxy-cytosines to the 3′ end of the cDNA with high efficiency. In a preferred embodiment a controlled ribonucleotide tailing is carried out with the aid of terminal transferase following the first strand cDNA synthesis.
Claims16
A method for the modification, cloning or amplification of a cDNA, wherein the first strand cDNA synthesis is carried out in the presence of magnesium2+ ions and manganese2+ ions.
A method for the modification, cloning or amplification of a cDNA, wherein the cDNA is incubated in the presence of manganese2+ ions directly after the first strand cDNA synthesis.
A mixture of double-stranded DNA adaptors containing 3′ overhangs composed of 5′-dT3-4dG3-3′.
First strand cDNA characterized by a non mRNA template-coded 3′ end composed of 5′-dC3-4rA3-4-3′.
Dependencies are inferred from the claim text.