LEQ: HOW DO WE SPLICE NEW GENES INTO DNA? 12.1 to 12.7 and 12.18

RECOMBINANT DNA TECHNOLOGY  This is a set of lab techniques for combining genes from different sources – even different species – into a single DNA molecule  Began with the study of Eschericia coli (E. coli)

TERMS FOR RECOMBINANT DNA TECHNOLOGY  Gene Cloning – the production of multiple copies of a gene  Genetic Engineering – the direct manipulation of genes for particle purposes  Biotechnology – the use of living organism (often microbes) to perform useful tasks

RESTRICTION ENZYMES  Bacterial enzymes that cut foreign DNA; original purpose was a defense mechanism of bacterial to protect against foreign DNA (phage DNA)  Restriction enzymes are now used to cut DNA molecules in reproducible ways  These enzymes produce 2 different kinds of ends 1. Blunt ends – these enzymes cut straight through the double strand of DNA; produces restriction fragments with no overlapping single strands; fragments cannot bond with other pieces of DNA 2. Sticky ends – these enzymes have a staggered cut resulting in fragments with single stranded ends which can bond with complementary DNA

STICKY OR BLUNT? StickyBlunt

Plasmids small circular ring of DNA found in prokaryotes and yeast Important sites of plasmid 1. origin of replication 2. genetic marker (i.e. antibiotic resistance) 3. restriction enzyme cut sites

Restriction Enzymes & Recombinant DNA Restriction enzymes have specific recognition sites Scientists use a specific enzyme to cut out a specific gene and a specific plasmid The sticky ends of the gene and the plasmid will compliment each other allowing scientists to join the two Use DNA Ligase to join two DNA types to produce recombinant DNA

Cloning Recombinant DNA 1. Identify a restriction enzyme that will cut out gene of interest and cut open the plasmid 2. Isolate DNA from 2 sources (making sure the at the plasmid has a genetic marker) 3. Cut both types of DNA with the same restriction enzyme 4. Mix the 2 types of DNA to join them through complementary base pairing Add 5. DNA ligase to bond DNA covalently producing Recombinant DNA 6. Incubate bacteria at 42 C with calcium chloride; bacteria become competent / permeable - so that the bacteria will take in the plasmid (TRANSFORMATION) 7. Use a genetic marker to identify bacteria with the recombinant plasmid 8. Clone bacteria

Clone Storage via Genomic Library Plasmid Libraries and Phage Libraries are created by cutting a genome into fragments Genome fragments are used to create recombinant DNA Recombinant DNA is then either stored in a bacterial culture or a phage culture

Reverse Transcriptase used to make genes for cloning 1. Transcribe DNA into RNA in the nucleus 2. RNA splicing occurs – removing introns 3. Isolate mRNA from the cell and add reverse transcriptase to synthesize a new strand of DNA 4. The mRNA is digested 5. Synthesize 2 nd complimentary strand using DNA polymerase creating “cDNA”

What’s the difference? DNA cDNA  Contains introns; - must be edited to be get to the DNA that codes for a gene  No introns – the actual DNA that codes for a particular gene

Genetically Modified Organisms Genetically modified organism – an organism that acquires one or more genes by artificial means (gene may or may not be from a different species) Transgenic organism – organism that contains a gene from another species

Recombinant DNA Applications Bacteria are protein factories. E. coli is the primary bacteria used; cheap and easy to maintain cultures and produce proteins (pharmaceutical factories) Products: Chymosin (used for cheese production), Human Insulin; Human Growth Hormone; Factor VIII; Hepatitis B vaccine; Diagnosis of HIV

Recombinant DNA Applications The yeast Saccharomyces cerevisiae is commonly used when eukaryotic cells are needed. Yeast serve as a good vector for human genomic libraries

Recombinant DNA Applications Plants By genetically modifying the genome of a plant scientists have been able to increase the nutritional value of crops (i.e. “Golden Rice” contains daffodil gene allowing it to produce beta-carotene); scientists have been able to genetically modify plants that are drought resistant, pesticide resistant, larger in size, and that have a longer shelf life.

Recombinant DNA Applications Mammals Recombinant DNA technology is used to add a human gene for a desired human trait (protein) to the genome of a mammal in such a way that the gene’s products, such as antithrombin (protein that prevents blood clots), are secreted in the milk of the animal; Transgenic mammals allow scientists to model human diseases and find treatments to the diseases; Transgenic pigs may serve as human blood and organ donors; Transgenic cattle & fish have been engineered to be larger in size – providing more meat

DNA Technology – pharmaceuticals and Medicine  Therapeutic Hormones – human insulin produced by bacteria (no longer use insulin from cadavers or pigs)  Diagnosis & Treatment of disease – identify disease causing alleles & tailored treatments  Vaccines – create harmless variants of pathogen to stimulate the immune system