Recombinant DNA Technology PDF

Recombinant DNA Technology Dr. Mona Rady rDNA Technology Intended learning outcomes: Define recombinant DNA technology and explain how it is used to clone genes and manipulate DNA. Identify the basic tools for a gene cloning experiment. Identify the properties of a good vector. The...

Recombinant DNA Technology Dr. Mona Rady rDNA Technology Intended learning outcomes: Define recombinant DNA technology and explain how it is used to clone genes and manipulate DNA. Identify the basic tools for a gene cloning experiment. Identify the properties of a good vector. The story of insulin the first human therapeutic protein approved by the FDA. GIU WS2022 2 Recombinant DNA technology Basic definitions Cutting and pasting DNA from different sources. rDNA technology made gene cloning possible. Genetic engineering relies on recombinant DNA technology and gene cloning to modify an organism’s genome. Clone is derived from a Greek word that describes a cutting (of a twig) that is used to propagate or copy a plant. A modern biological definition of a clone is a molecule, cell, or organism produced from another single entity. GIU WS2022 3 The first recombinant DNA in history! Paul Berg created a piece of recombinant DNA by joining together (splicing) DNA from the E. coli chromosome and DNA from a primate virus called SV40. 1980 Nobel Prize in Chemistry for this experiment, which demonstrated that DNA could be cut from different sources with the same enzyme and that the restriction fragments could be joined to create a recombinant DNA Paul Berg Stanford University, Stanford, CA, USA molecule. GIU WS2022 4 rDNA technology – Historical background In 1975, an invited group of well-known molecular biologists, virologists, microbiologists, lawyers, and journalists gathered in California, to discuss the benefits and potential hazards of recombinant DNA technology. As a result of the historic meeting, the National Institutes of Health (NIH) formed the Recombinant DNA Advisory Committee (RAC), which was charged with evaluating the risks of recombinant DNA technology and establishing guidelines for recombinant DNA research. In 1976, the RAC published a set of guidelines for working with recombinant organisms. The RAC continues to oversee gene cloning research, and compliance with RAC guidelines is mandatory for scientists working with recombinant organisms. GIU WS2022 5 What made rDNA technology possible? Restriction enzymes (restriction endonucleases) Plasmids (plasmid DNA)/vectors DNA ligase enzyme Host cell (accepts rDNA and potentially expressing the recombinant protein) Method for introducing rDNA into host cell DNA cloned into a plasmid/vector is called “insert” DNA. GIU WS2022 6 Restriction enzymes/restriction endonucleases Bacteriophage infecting a DNA-cutting enzymes. bacterial cell Bacteriophage “scissors” needed to carry out gene DNA is Restriction cloning. enzyme(s) fragmented Used by bacteria to destroy bacteriophages. They can “restrict” phage replication. Also called restriction endonucleases (endo, “within”; nuclease, “nucleic acid–cutting enzyme”) because they cut within DNA sequences as opposed to enzymes that cut from the ends of DNA sequences (exonucleases). GIU WS2022 7 Structure of DNA GIU WS2022 8 Restriction enzymes Why don’t restriction enzymes digest DNA in bacterial cells? Cut DNA by cleaving the phosphodiester bond (in the sugar-phosphate backbone) that joins adjacent nucleotides in a DNA strand. Bind to, recognize, and cut (digest) DNA within specific sequences of bases called restriction sites. Bacteria protect their DNA from restriction enzyme digestion because some of the nucleotides in their DNA contain methyl groups that block restriction enzymes from digestion. GIU WS2022 9  Restriction enzymes Four- or eight-base-pair cutters Recognize restriction sites with a sequence of 4 or 8 nucleotides. Each restriction site is a palindrome; the arrangement of nucleotides reads the same forward and backward on opposite strands of the DNA molecule. GIU WS2022 10 Restriction enzymes Some restriction enzymes cut DNA to create DNA fragments with overhanging single-stranded ends called “sticky” or cohesive ends. Other enzymes generate fragments with double-stranded ends called blunt ends. Enzymes that produce cohesive ends are often favored over blunt-end cutters for many cloning experiments because DNA fragments with cohesive ends can easily be joined together. GIU WS2022 11 Naming Restriction enzymes They are given abbreviated names based on the genus and species names of the bacteria from which they are isolated. Example: EcoRI E = genus Escherichia co= species coli R = strain RY13 I = First endonuclease identified in E. coli. GIU WS2022 12 Types of restriction enzymes Three major classes Type I and III restriction enzymes bind to the DNA at their recognition sequences but cut the DNA at some distance away. Type II restriction enzymes cleave the DNA within their recognition sequences. Type II restriction enzymes are more useful for the specific manipulation of DNA. GIU WS2022 13 Plasmids Extrachromosomal DNA because they are present in the bacterial cytoplasm in addition to the bacterial chromosome. Plasmids are small; most average approximately 1,000 to 4,000 base pairs (bp) in size. Self-replicating; that is, they duplicate independently of the chromosome. Plasmids could be used as vectors— pieces of DNA that can accept, carry, and replicate (clone) other pieces of DNA. DNA ligase catalyzes the formation of phosphodiester bonds between nucleotides. Ligase can join together DNA with cohesive ends as well as blunt ended fragments. GIU WS2022 14 Creating Recombinant DNA GIU WS2022 15 A Typical Genetic Modification Procedure § Genes from one organism’s cells can be inserted and expressed in another organism’s cells. § Genetically modified cells can be used to create a wide variety of useful products and applications. GIU WS2022 16 Bacterial transformation A process for inserting foreign DNA into bacteria (the host cell). Can be done by treating bacterial cells with calcium chloride (positively charged chemical; WHY?) solutions, added plasmids to cells chilled on ice, and then briefly heated the cell and DNA mixture, plasmids entered bacterial cells (Competency). Electroporation, involves applying a brief (millisecond) pulse of high- voltage electricity to create tiny holes in the bacterial cell wall that allow DNA to enter. Once inside bacteria, plasmids replicate and express their genes. GIU WS2022 17 Transformation of Bacterial Cells by Calcium Chloride Treatment GIU WS2022 18 Electroporation Is a Rapid and Effective Technique for Transforming Bacteria GIU WS2022 19 What are bacterial colonies? Bacteria grow on solid media as colonies. A colony is defined as a visible mass of microorganisms all originating from a single mother cell. A colony constitutes a clone of bacteria all genetically alike. GIU WS2022 20 Selection of recombinant bacteria Why do we need to select recombinant bacteria? 1. During ligation, some of the digested plasmid ligates back to itself to create recircularized plasmid that lacks foreign DNA. 2. During transformation, a majority of cells do not take up DNA. Selection of recombinant bacteria aims to the identification of (selecting for ) recombinant bacteria while preventing the growth of (selecting against ) nontransformed bacteria and bacteria that contain plasmid without foreign DNA. GIU WS2022 21 Antibiotic selection of recombinant bacteria A technique in which transformed bacterial cells are plated on agar plates (nutrient medium for growth of bacteria) with different antibiotics, as a way to identify recombinant bacteria (carry antibiotic resistance gene(s)) and nontransformed cells. GIU WS2022 22  “Blue-white” selection of recombinant bacteria DNA is cloned into a restriction site in the lacZ gene. lacZ gene encodes β- galactosidase (β-gal), an enzyme that degrades the disaccharide lactose into the monosaccharides glucose and galactose. When it is interrupted by an inserted gene, the lacZ gene is incapable of producing functional β-gal. GIU WS2022 23 Cloning a Gene in a Plasmid and Blue-White Selection Blue colonie Transformed with non-recombinant plasmid White colonie Transformed with recombinant plasmid GIU WS2022 24  Cloning a Gene in a Plasmid and Blue-White Selection Transformed bacteria are plated on agar plates that contain an antibiotic (ampicillin). Nontransformed bacteria cannot grow in the presence of ampicillin because they lack plasmids containing an ampicillin resistance gene (ampR). Antibiotic selection alone does not distinguish transformed bacteria with nonrecombinant plasmid that has recircularized from recombinant plasmids. GIU WS2022 25  Cloning a Gene in a Plasmid and Blue-White Selection To identify bacteria with recombinant plasmids, the agar must also contain a chromogenic (color-producing) substrate for β-gal called X- gal. X-gal is similar to lactose in structure and turns blue when cleaved by β-gal. Nonrecombinant bacteria—those that contain plasmid that ligated back to itself without insert DNA—contain a functional lacZ gene, produce β-gal, and turn blue. Conversely, recombinant bacteria are identified as white colonies. Because these cells contain plasmid with foreign DNA inserted into the lacZ gene, β-gal is not produced, and these cells cannot metabolize X-gal (white colonies). Colonies containing recombinant plasmids are clones— genetically identical bacterial cells each containing copies of the recombinant plasmids. GIU WS2022 26 Human gene cloning; the story of recombinant insulin If the cloned DNA fragment is a gene that encodes a protein product, bacterial cells could be used to synthesize the protein product of the cloned gene. We call this “expressing” a protein. Human genes can be cloned and expressed in bacterial cells. Because bacteria can be grown in large-scale preparations, scientists can produce large amounts of the cloned DNA and isolate quantities of protein that would normally be very difficult or expensive to purify without cloning. The first commercially available human gene product of recombinant DNA technology was human insulin hormone. GIU WS2022 27 Human gene cloning; the story of recombinant insulin GIU WS2022 28 Human gene cloning; the story of recombinant insulin In 1977, the insulin gene was cloned into plasmids, expressed in bacterial cells, and isolated by scientists at Genentech (named for genetic engineering technology). Genentech is the first biotechnology company established in San Francisco. In 1982, the recombinant form of human insulin, called Humulin, became the first recombinant DNA product to be approved for human applications by the U.S. Food and Drug Administration (FDA). GIU WS2022 29 Blue-white screening for selecting recombinant bacteria Remember that! § Antibiotic selection selects cells that are transformed with recombinant and non- recombinant plasmids. § Blue white selection differentiates beween bacteria transformed with recombinant versus non-recombinant plasmids. § β-galactosidase gene (lacZ) codes for β- galactosidase enzyme that breaks X-gal and gives a blue color. § Blue colonies are cells having functional lacZ gene; transformed with non-recombinant plasmids why?? GIU WS2022 30 Thank You GIU WS2022 31