5' protection dependent amplification

摘要

The present invention relates to methods for generating a labelled nucleic acid from an RNA comprising a 5′ protecting group, said method comprises the steps of obtaining a mixture of template strands of nucleic acids, said mixture comprising said RNA and further potentially other nucleic acids without a 5′ protecting group, annealing at least one oligonucleotide primer to the template strand of said RNA and potentially other nucleic acids, and template sequence dependent extending said primer, thereby obtaining a complementary nucleic acid strand annealed to its template strand, or providing the RNA in duplex with a complementary nucleic acid strand annealed to its template strand, and;optionally modifying the extension product of said nucleic acids without 5′ protecting group either on the 5′ end of the template strand or on the 3′ end of the complementary strand, or both, and labelling a complementary nucleic acid of a double stranded nucleic acid not modified, wherein therefore the labelled nucleic acid did have a 5′ protecting group on the RNA template strand, said 5′ protecting group being optionally removed after modification, and/or labelling a complementary nucleic acid of a double strand dependent on the presence of the 5′ protecting group on the complementary nucleic acids template RNA strand by double strand dependent ligation, thereby specifically generating a labelled nucleic acid complementary to an RNA at least originally comprising a 5′ protecting group.

主权项

1. A method for generating a labelled nucleic acid from an RNA comprising a 5′ protecting group, preferably said protecting group being a 5′ cap or a 5′ polyphosphate, said method comprises the steps of:
a) obtaining a mixture of template strands of nucleic acids, said mixture comprising said RNA and further potentially other nucleic acids without a 5′ protecting group, b1) annealing at least one oligonucleotide primer to the template strand of said RNA and potentially other nucleic acids, and template sequence dependent extending said primer, thereby obtaining a complementary nucleic acid strand annealed to its template strand, or b2) providing the RNA in duplex with a complementary nucleic acid strand annealed to its template strand, and c) optionally modifying the extension product of said nucleic acids without 5′ protecting group either on the 5′ end of the template strand or on the 3′ end of the complementary strand, or both, and d1) labelling a complementary nucleic acid of a double stranded nucleic acid not modified in step c), wherein therefore the labelled nucleic acid did have a 5′ protecting group on the RNA template strand in step c), said 5′ protecting group being optionally removed after performing step c), and/or d2) labelling a complementary nucleic acid of a double strand dependent on the presence of the 5′ protecting group on the complementary nucleic acids template RNA strand by double strand dependent ligation, thereby specifically generating a labelled nucleic acid complementary to an RNA at least originally comprising a 5′ protecting group.