Enzymes Used In Recombinant DNA Technology PDF

Enzymes used in Recombinant DNA Technology Dr. N.M.C. Nayanakantha Seniour Lecturer (Grade 1)  1. Restriction Enzymes A restriction enzyme is a nuclease enzyme that cleaves DNA sequence at a random or specific recognition sites known as res...

Enzymes used in Recombinant DNA Technology Dr. N.M.C. Nayanakantha Seniour Lecturer (Grade 1)  1. Restriction Enzymes A restriction enzyme is a nuclease enzyme that cleaves DNA sequence at a random or specific recognition sites known as restriction sites. In bacteria, restriction enzymes form a combined system (restriction + modification system) with modification enzymes that methylate the bacterial DNA. Methylation of bacterial DNA at the recognition sequence typically protects the own DNA of the bacteria from being cleaved by restriction enzyme. There are two different kinds of restriction enzymes: (1) Exonucleases catalyses hydrolysis of terminal nucleotides from the end of DNA or RNA molecule either 5’to 3’ direction or 3’ to 5’ direction. Example: exonuclease I, exonuclease II. (2) Endonucleases can recognize specific base sequence (restriction site) within DNA or RNA molecule and cleave internal phosphodiester bonds within a DNA molecule. Example: EcoRI, Hind III, BamHI. History of Restriction Enzymes In 1970 the first restriction endonuclease enzyme HindII was isolated. In 1978 Daniel Nathans, Werner Arber, and Hamilton O. Smith awarded for Nobel Prize for Physiology or Medicine for the subsequent discovery and characterization of numerous restriction endonucleases. Since then, restriction enzymes have been used as an essential tool in recombinant DNA technology. Restriction enzymes nomenclature: Restriction endonucleases are named according to the organism in which they were discovered, using a system of letters and numbers. For example, HindIII (pronounced “hindee-three”) was discovered in Haemophilus influenza (strain d). The Roman numerals are used to identify specific enzymes from bacteria that contain multiple restriction enzymes indicating the order in which restriction enzymes were discovered in a particular strain. Classification of Restriction Endonucleases: There are three major classes of restriction endonucleases based on the types of sequences recognized, the nature of the cut made in the DNA, and the enzyme structure: Type I restriction enzymes Type II restriction enzymes Type III restriction enzymes Type II restriction enzymes: Cleavage of nucleotide sequence occurs at the restriction site. These enzymes are used to recognize rotationally symmetrical sequence which is often referred as palindromic sequence. They require only Mg2+ as a cofactor and ATP is not needed for their activity. Type II endonucleases are widely used for mapping and reconstructing DNA in vitro because they recognize specific sites and cleave. Cleavage Patterns of Some Common Restriction Endonucleases: The recognition and cleavage sites and cleavage patterns of HindIII, SmaI, EcoRI, and BamHI are shown. Cleavage by an endonuclease creates DNA sequence with either a sticky end or blunt end. The blunt ended fragments can be joined to any other DNA fragment with blunt ends using linkers/adapters, making these enzymes useful for certain types of DNA cloning experiments. Applications of Restriction Enzymes: In various applications related to genetic engineering DNA is cleaved by using these restriction enzymes. They are used in the process of insertion of genes into plasmid vectors during gene cloning and protein expression experiments. Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms (SNPs). This is only possible if a mutation alters the restriction site present in the allele. Restriction enzymes are used for Restriction Fragment Length Polymorphism (RFLP) analysis for identifying individuals or strains of a particular species.  2. Deoxyribonuclease (DNase): Unlike restriction enzymes, DNase does not have any specific recognition/restriction site and cleave DNA sequence at random locations. 3. Ligases DNA ligase catalyses the formation of phosphodiester bond between two deoxynucleotide residues of two DNA strands. DNA ligase enzyme requires a free hydroxyl group at the 3´ -end of one DNA chain and a phosphate group at the 5´-end of the other and requires energy in the process. E.coli and other bacterial DNA ligase utilizes NAD+ as energy donor, whereas in T4 bacteriophage, T4 DNA ligase uses ATP as cofactor. The role of DNA ligase is to seal nicks in the backbone of double-stranded DNA after lagging strand formation to join the Okazaki fragments. This joining process is essential for the normal synthesis of DNA and for repairing damaged DNA. Eg. T4 DNA Ligase  Applications of ligase enzyme It has been exploited by genetic engineers to join DNA chains to form recombinant DNA molecules. DNA ligase enzyme is used by cells to join the “okazaki fragments” during DNA replication process. In molecular cloning, ligase enzyme has been routinely used to construct a recombinant DNA. Joining of adapters and linkers to blunt end DNA molecule. Cloning of restricted DNA to vector to construct recombinant vector. DNA replication and formation of Okazaki fragments 4. Ribonuclease (RNase): Nuclease that can catalyze hydrolysis of ribonucleotides from either single stranded or double stranded RNA sequence are called ribonucleotides (RNase) Application: It is used to remove RNA contamination from DNA sample.