6 Introduction Enzymes are the keys of success of genetic engineering. Along with restriction enzymes which functions in the cleavage of DNA there are certain enzymes required to rebind it.These enzymes join the DNA with vector & helps in the formation of recombinant DNA.
7 S1 NucleaseS1 nuclease is an endonuclease active against single-stranded DNA and RNA molecules.It is five times more active on DNA than RNA.Its reaction products are oligonucleotides or single nucleotides with 5' phosphoryl groups.Its primary substrate is single-stranded, but also introduce single-stranded breaks in double-stranded DNA or RNA, or DNA-RNA hybrids.
9 Application of S1 Nuclease It is used in the laboratory as a reagent in nuclease protection assays.In Molecular biology it is used in removing single stranded tails from DNA molecules to create blunt ended molecules and opening hairpin loops generated during synthesis of double stranded cDNA.
10 DNA LigaseDNA ligases are vital enzymes required for important cellular processes such as DNA replication, repair of damaged DNA and recombination.The enzyme mediates the formation of phosphodiester bonds between adjacent 3'-OH and 5'-phosphate termini, thereby joining the nicks in double stranded DNA.
11 Types of DNA LigasesLigases can be classified into two groups depending on their requirement for ATP or NAD+ as the cofactor.All eukaryotic and virally encoded enzymes are ATP-dependent.Example- DNA ligase from bacteriophage T7.Most prokaryotic enzymes require NAD+ for their activity.Example- DNA ligase from B.stearothermophilus.In mammals, four types of DNA ligases are present.
12 T4 DNA LigaseIt is used for covalently joining restriction fragments along with E.coli DNA ligase.E.coli DNA ligase uses NAD+ while T4 DNA ligase requires ATP.T4 DNA ligase has the ability to join the blunt ends of DNA fragment, whereas E.coli DNA ligase joins the cohesive ends produced by restriction enzymes.T4 enzyme can quickly join & produce the full base pairs.
13 DNA Ligase In Molecular Biology DNA ligase has extensive use in molecular biology laboratories for that can link together DNA strands that have double-strand breaks.
14 Applications of DNA Ligase DNA ligases are used with restriction enzymes to insert DNA fragments, often genes, into plasmids.To perform successful recombination experiments ligase is use for controlling the optimal temperature.Most experiments use T4 DNA Ligase which is most active at 25°C.In order to perform successful ligations, the optimal enzyme temperature needs to be balanced with the melting temperature Tm & the annealing temperature of the DNA fragments being ligated.
15 What We Learn………….S1 nuclease can introduce break in single stranded DNA or RNA.It acts intensively on DNA than RNA & create blunt ends.DNA ligase carry out three types of actions repair, replication & recombination.In molecular biology, their vital role is the ligation of DNA into vector.