LF-111 Lecture (6) Recombinant DNA Technology Lecture Notes PDF

LF111 Lecture (6-7) 25-26 October_ 2024 Recombinant DNA technology Genetic Engineering  Background: Meaning of the term “Clone” in biology In bacteria individual colonies represents identical copies of single mother cells. Thus each colony represents a clone Can there be clones in hi...

LF111 Lecture (6-7) 25-26 October_ 2024 Recombinant DNA technology Genetic Engineering  Background: Meaning of the term “Clone” in biology In bacteria individual colonies represents identical copies of single mother cells. Thus each colony represents a clone Can there be clones in higher organisms? Why do we use the term DNA clone in genetic engineering?  Recap: Griffith’s experiment Bacterial transformation What does transformation signify? What opportunities does this An incoming bacterial factor (DNA ) observation offer? could replace an endogenous factor Could an incoming DNA ( foreign gene) be made to stay in a cell?  Some background information ▪ Prokaryotes are haploid: for example, bacteria E. coli – it has only one chromosome ▪ Bacterial chromosome is circular ▪ Bacterial chromosome represent a single unit of replication Bacterial chromosome Plasmids ▪ Bacterial cell can carry extra chromosomal replicating bodies, called plasmids  Transformation Transformation essentially means What are the continuation of a piece of barriers to DNA or gene in a host for continuity of an incoming generations. DNA in a cell? Thus, the DNA or the gene is said to be “Cloned” What is the advantage of recombination here? Incoming DNA Answer: must replicate Incoming DNA now to continue for generations replicates with the rest of the bacterial genome Is this genetic engineering? Answer: No, it happens naturally Transformation by a plasmid (vector) DNA Host bacterium Host bacterium Transformed by a plasmid The transformed host bacterium and it progeny would express the gene (marker) carried by the plasmid: for instance, antibiotic resistance What if the plasmid (vector) DNA is made into a recombinant DNA first before transformation?  Definitions of recombinant DNA (rDNA) and genetic engineering ▪ Recombinant DNA: A new DNA molecule created by joining together two or more DNA fragments not normally associated with each other: for example a DNA from human and bacteria ▪ Genetic engineering: The end product of genetic engineering is an organism that has been genetically modified using recombinant DNA technology without taking recourse to its natural course of mutation/ evolutionary changes etc Recombinant DNA: definition A unique piece of DNA artificially created by joining them together Recombinant DNA: Vocabulary ▪ Vector ▪ Recombinant DNA ▪ Restriction enzyme ▪ Ligase ▪ Transformation ▪ Selection ▪ Gene clone  Cloning Vectors ▪ A cloning vector is a DNA molecule that has an origin of replication and is capable of replicating in a bacterial cell. ▪ Most vectors are genetically engineered plasmids or phages.  Cloning vector What is a replicon? Plasmids Plasmids: Extra-chromosomal genetic elements that are capable of autonomous replication (replicon) What does these green area on the plasmids signify? What are the A marker (gene) that can be advantages of readily detected – antibiotic having a cloning resistance, for example vector Transformation Step 1 Step2 Characteristics of plasmid cloning vector : ▪ Small size (