Genetic Engineering Intent of altering human genome Introducing new genetic material into genome Isolating genes to produce on large scale (Insulin)

Recombinant DNA DNA that contains genes of two species How? Restriction enzymes – cut out desired gene Occur naturally in prokaryotic cells Recognize specific recognition sites – 4 to 8 base pairs Recognition sites are palindromes Cuts gene (digests) in one direction only Creates restriction fragments

Process – Restriction Enzyme Locates recognition site (Top Strand) Cuts the DNA backbone Locates recognition site (Bottom Strand) DNA separates

Sticky Ends or Blunt Ends Sticky ends – zigzag cuts in strand Blunt ends – straight cut across strand

Putting Fragments together DNA ligase – sticky ends T4 DNA ligase – blunt ends forms phosphodiester bonds in DNA

Plasmids Small circular pieces of DNA found in bacteria Used as vectors for recombinant DNA (artificial) Restriction enzymes used to isolate specific gene are used to cut plasmids

Process Plasmids and DNA fragments are placed in same solution Anneal DNA ligase is used to form phosphdiester bond Recombinant DNA introduced into host cell DNA is cloned

Identifying Bacteria Clones with Target Genes Hybridization – identify cells that contain recombinant DNA Identified using a hybridization probe – short single stranded complementary DNA molecule Once identified bacteria can be grown in huge quantities (commercial use)

Transformation Cells that receive foreign DNA Bacterial cells sometimes will not take up a plasmid Bacteria are placed in ice water bath containing CaCl₂ Solution is heated and cooled repeatedly disrupting plasma membrane of bacteria allowing plasmid to enter Solution is kept at 37⁰C to stabilize and grow

Cloning Insulin

Restriction Maps Diagrams that show all recognition sites on a specific plasmid and distances in base pairs Shows which restriction enzyme should be used Allow scientists to determine which plasmids will work the best for cloning experiments

Gel Electrophoresis Technique used to separate fragments of DNA (PCR) Used to help identify information about unknown pieces of DNA (restriction maps/forensics)

PCR Polymerase Chain Reaction Process Increase number of DNA copies from a single biological sample in a few hours Only specific regions of a chromosome are replicated Process Denaturation Annealing Elongation

Taq polymerase is used to put strand together Isolated from bacteria that live in hot springs

Genetic Engineering Biopharming Pharmaceutical products produced on large scale Organisms are genetically engineered to produce a specific protein Ability to make new protein is passed on to offspring

Genetic Engineering Transgenic Organism (genetically modified organism, GMO) Organisms that contain one or more genes from another organism

Knockout Mice

Why use Genetic Engineering? Cost Larger organisms can produce larger molecules Better versions of organisms 80% of Canadas Canola crop is GM

Gene Therapy Techniques used to replace, remove or alter a defective gene before symptoms are expressed Insert healthy genes into cells of tissues that are affected by a disorder

Gene Therapy

Gene Therapy Germ-line gene therapy Somatic gene therapy Genes introduced in sperm or egg cells Passed on to future generations Somatic gene therapy Genes introduced into body cells (not sperm or egg) Will not be passed on

Biotechnology Artificial Insemination In Vitro Fertilization Sperm is collected and concentrated before being placed in woman’s uterus In Vitro Fertilization Eggs from mom are retrieved and combined with sperm in petri dish After fertilization, zygote is placed in woman’s uterus

Genetic Testing Determining if your baby carries a gene that is associated with an increased risk of developing a disease

Aminocentesis Benefits Risks Diagnosis of chromosome abnormalities Discomfort for expectant mother Slight risk of infection Diagnosis of other defects Miscarriage

Chorionic Villus Sampling Benefits Risks Diagnosis of chromosome abnormalities Miscarriage Infection Newborn having limb defects Diagnosis of certain genetic defects

Embryonic Stem Cells – differentiate into any kind of cell Stem Cell – an undifferentiated cell that can divide to form specialized cells Embryonic Stem Cells – differentiate into any kind of cell Muscle Cell Skin Cell Nerve Cell Heart Cell Tissue Stem Cells (Adult Stem Cells) – exist within specialized tissue, differentiate into certain types of cell s Bone Marrow Stem Cells

Cord Blood Banking Source of hematopoietic (blood forming) stem cells Blood (stem cells) found in umbilical cord after birth Similar to tissue stem cells Develop into various blood cells These stem cells could be banked (stored) Used to treat variety of diseases

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