RNase H is a 18.9 kDa recombinant endoribonuclease purified from an Escherichia coli strain. The enzyme hydrolyses specifically the phosphodiester bonds of RNA hybridized to DNA and produces 5´ phosphate-terminated oligoribonucleotides and single-stranded DNA. The enzymes does not degrade single and double-stranded DNA or unhybridized RNA. It is a key enzyme in the removal of mRNA after first-strand cDNA synthesis. Treating cDNA with RNase H prior to PCR can improve sensitivity as RNA bonded to the cDNA template may prevent binding of the amplification primers in a PCR reaction. RNase treatment is often necessary when amplifying longer, full-length cDNA targets. In addition, this enzymes is useful for the removal of poly(A) tails on mRNAs after hybridization with oligo(dT) and also for the site-specific enzymatic cleavage of RNA.
Tth DNA Ligase catalyzes the NAD-dependent formation of phosphodiester bonds between adjacent 3’-hydroxyl and 5’-phosphate termini in double stranded DNA. It is not active against single stranded DNA or RNA and blunt ended DNA.