Protocol for the generation and purification of high-molecular-weight covalent RNA-DNA hybrids with T4 RNA ligase.

ABSTRACT

RNA-DNA covalent hybrids (RDHs) are widely employed in biology. Although RDHs can be manufactured, the synthesis of molecules longer than 120 nucleotides is challenging. Here, we present a protocol for the generation and purification of high-grade purified high-molecular-weight 5'-RNA-DNA-3' hybrids. We describe steps for preparing oligos and buffers, ligation reaction, and high-performance liquid chromatography-based RDH purification. This protocol is executable in standard molecular biology laboratories.