DNA/RNA Synthesis

RNA synthesis, which is also called DNA transcription, is a highly selective process. Transcription by RNA polymerase II extends beyond RNA synthesis, towards a more active role in mRNA maturation, surveillance and export to the cytoplasm.

Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template, with DNA ligase creating the final phosphodiester bond to fully repair the DNA.DNA ligases discriminate against substrates containing RNA strands or mismatched base pairs at positions near the ends of the nickedDNA. Bleomycin (BLM) exerts its genotoxicity by generating free radicals, whichattack C-4′ in the deoxyribose backbone of DNA, leading to opening of the ribose ring and strand breakage; it is an S-independentradiomimetic agent that causes double-strand breaks in DNA.

First strand cDNA is synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H). Second strand cDNA synthesis is subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs are converted into blunt ends using exonuclease/polymerase activity. After adenylation of the 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare the samples for hybridization. Cell cycle and DNA replication are the top two pathways regulated by BET bromodomain inhibition. Cycloheximide blocks the translation of mRNA to protein.