Biotechnology Biotechnology is the use of biological processes, organisms, or systems to manufacture products intended to improve the quality of human life.
Genetic Engineering- (A.K.A. Recombinant DNA Technology) • frequency of an allele in a population • *involves cutting (cleaving) DNA from one organism into small fragments & inserting the fragments into a host organism of the same or a different species
AMAZING!!! Organism will use the foreign DNA as if it were its own!! • Transgenic Organism- organisms that contain functional recombinant DNA (rDNA) from a different organism
4 Areas of Biotechnology • Agriculture • Industry • Forensics • Medicine
Remember DNA? • What is the monomer of DNA? • Nucleotides • How do bases pair? • A – T • C – G • What kind of bond is used? • Hydrogen bonds between nitrogen bases
I. Restriction Enzymes • AKA Restriction Endonucleases • What macromolecule do you think they are made of? • They are PROTEINS that cut strands of DNA at specific nucleotide sequences
Isolating foreign DNA fragments • -Restriction Enzymes- DNA cutting enzymes that can cut both strands of a DNA molecule at a specific base pair sequence (A-T, C-G) • -similar to cutting a zipper into pieces • -must find the same sequence of base pairs on both DNA strands but they must run in opposite directions
Restriction Enzymes (cont.) • There are many different restriction enzymes that each cut DNA at different nucleotide sequences • Most will cut the DNA with a staggered cut • Usually occurs at a palindrome: a sequence of units that can be read the same way in either direction (ex. Mom, dad, racecar) 5‘…GAATTC…3’ 3‘…CTTAAG…5’
Sticky Ends • The staggered cuts leave the DNA with end pieces “sticking off” • We call these “sticky ends” • These exposed N-bases will want to join with other complimentary exposed bases
E. Types of Restriction Enzymes • Sticky End- already discussed • Blunt End • These cut the DNA straight across and create blunt ends: CCC GGG GGG CCC
F. Products generated by restriction enzymes 1. COHESIVE END CUTTERS (staggered cuts): EnzymeRecognition SiteEnds of DNA After Cut 5’…G AATTC…3’ 3’…CTTAA G…5’ 5’…GAATTC…3’ 3’…CTTAAG…5’ EcoRI 5’…CTGCA G…3’ 3’…G ACGTC…5’ Pst I 5’…CTGCAG…3’ 3’…GACGTC…5’ 2. BLUNT END CUTTERS (direct cuts): EnzymeRecognition SiteEnds of DNA After Cut 5’…GG CC…3’ 3’…CC GG…5’ 5’…GGCC…3’ 3’…CCGG…5’ HaeIII
G. Restriction Enzyme Naming 1. Restriction enzymes are named according to the following nomenclature: Ex: EcoRI • E = genus Escherichia • co = species coli • R = strain RY13 • I = first enzyme isolated
How is a transgenic organism formed?? • Isolate foreign DNA fragment • Attach DNA fragment to a “vehicle” (vector) • Transfer “vehicle” (vector) into a host organism
Why would anyone go through the trouble of cutting DNA??? • One reason… • Recombinant DNA • Break down the word…what do you think recombinant means? • Other reasons… • DNA fingerprinting, gene therapy…
Recombinant DNA • Recombinant DNA: DNA that has been cut from one strand of DNA and then inserted into the gap of another piece of DNA that has been broken. • The host DNA is often a bacterial cell such as E coli.
Bacterial Structure • Bacteria are often used in biotechnology because they have plasmids • A PLASMID is a circular piece of DNA that exists apart from the chromosome and replicates independently of it. • A plasmid is therefore called a VECTOR.
Vectors transfer DNA • Vector-means by which DNA from another species can be carried into the host cell • Mechanical Vectors • Micropipette-inserts into a cell • Gene guns- tiny metal bullet is coated with DNA and shot into the cell with a gene gun
More types of Vectors • Biological Vectors • Viruses • Plasmids-small ring of DNA found in bacteria cells that is separate from the bacteria’s normal set of DNA • Plasmid usually contains genes that may cause the bacteria to be resistant to certain antibiotics
D. Isolating Genes • Must isolate the gene of interest first before you insert it into the plasmid • How do you do this? • Use a restriction enzyme!!!
Final Steps of Making Recombinant DNA • Once the gene is isolated, have to cut the organism’s DNA with the same restriction enzyme…why? • The sticky ends will naturally be attracted to each other • Add DNA LIGASE: enzyme that seals the fragments together • After the foreign DNA has been spliced (glued) into the plasmid using an enzyme DNA ligase, the rDNA is transferred into a bacterial cell or other organism • Now organism is called a Transgenic Organism- organisms that contain functional recombinant DNA (rDNA) from a different organism
Gene Splicing/Cloning using a bacterial plasmid • -IMPORTANT plasmid replicates separately from the bacterial chromosome & can produce up to 500 copies per bacterial cell • -bacteria reproduce quickly (20 min) so a lot of rDNA is made very fast • You will essentially be cloning a gene- genetically identical copies of rDNA molecules • -Host cell produces the protein coded for by the rDNA
III. Uses for Recombinant DNA • Recombinant DNA has been gaining importance over the last few years, and will become more important as genetic diseases become more prevalent and agricultural area is reduced. Below are some of the areas where Recombinant DNA will have an impact: • Better Crops (drought & heat resistance) • GMO’s (crops like seedless watermelon, pluots, etc.) • Recombinant Vaccines (i.e. Hepatitis B) • Production of clotting factors • Production of insulin • Production of recombinant pharmaceuticals • Plants that produce their own insecticides • Germ line and somatic gene therapy
RECAP • Steps for making a transgenic organism: • Locate and isolate the gene of interest • Cut out the gene and cut the plasmid using the appropriate restriction enzyme
5. Transfer the vector in the host organism where it will replicate 6. Host organism produces the protein coded for by the recombinant DNA
Gene Therapy • Gene therapy attempts to treat genetic diseases at the molecular level by correcting what is wrong with defective genes. • Clinical research into gene therapy’s safety and effectiveness has just begun. • No one knows if gene therapy will work, or for what diseases. If gene therapy is successful, it could work by preventing a protein from doing something that causes harm, restoring the normal function of a protein, giving proteins new functions, or enhancing the existing functions of proteins
Gene Therapy • In vivo gene therapy requires that the gene transfer vector be delivered by direct tissue injection. • 2) Ex-vivo gene therapy involves removing tissue from the patient, transfecting (or virally-infecting) the cells in culture, and then reimplanting the genetically altered cells to the patient.