Biotechnology Part I Genetic Engineering Recombinant DNA Purpose
Biotechnology Part I: Genetic Engineering
Recombinant DNA • Purpose: To insert genes of one organism into another organism
Recombinant DNA Applications • Green-glowing aquarium fish (jellyfish genes)
Recombinant DNA Applications • Fast-growing fish (Salmon with Pout genes)
Recombinant DNA Applications • Herbicide-resistant crops (Round-Up Ready)
Recombinant DNA Applications • Pest-resistant crops (Bt toxin)
Recombinant DNA Applications • Medicine production (bacteria can make insulin, h. GH, etc)
Making Recombinant Bacteria • Important players: • • Gene of interest (i. e. insulin, h. GH, etc) Restriction enzymes – cut the DNA Plasmid – circular DNA found in bacteria Bacteria
What are Restriction Enzymes? • Restriction enzymes: • • • Target very specific base sequences Are found in more than 100 different varieties Are used in nature to protect bacteria from foreign invaders
What are Restriction Enzymes? Each restriction enzyme recognizes a very specific nucleotide sequence Eco. R 1 recognizes: GAATTC CTTAAG The enzyme cuts it: G AATTC CTTAA G •
Making Recombinant DNA • Plasmids - selfreplicating rings of DNA containing 2 -30 genes, found in bacterial cells
Making Recombinant DNA 1. 2. 3. 4. 5. 6. Isolate the gene of interest using a restriction enzyme Cut the plasmid (using the same enzyme) Insert gene into the plasmid Insert the plasmid into bacteria Grow bacteria Harvest & purify the protein
1. Isolating the gene • Use restriction enzymes to cut the DNA strand at specific places that surround the gene
2. Cut the plasmid • Cut the plasmid using the same restriction enzyme
3. Put the gene in the plasmid • Since the gene and the plasmid have the same sticky ends, the gene will stick to the plasmid
5/6. Put plasmid in bacteria • • Insert plasmid into bacteria Let bacteria make the protein. Harvest protein & purifty it.
Biotechnology Part II: DNA Fingerprinting
DNA Fingerprinting • • Definition: Method of visualizing the differences in DNA samples Purpose: • • Forensic science Determining paternity Research Diagnosing Disease
Gel Electrophoresis • Separates DNA fragments by size using electric current
Gel Electrophoresis • • Larger fragments move more slowly Bands of fragments result
DNA Fingerprinting Method • Use restriction enzymes to cut the DNA • • • Unique DNA sequences produce unique banding patterns on the gel Load the DNA onto agarose gel for gel electrophoresis Analyze the banding pattern
Gel Electrophoresis – Paternity Test
Gel Electrophoresis – Crime investigation
RFLP Analysis RFLP – Restriction Fragment Length Polymorphism: • Restriction Enzymes used on two (or more) samples of DNA • Different DNA fragments are produced because of variation in the DNA sequences • Genetic markers are used for fingerprinting • Gene sequences are used for genetic tests
A single nucleotide change can make a difference Wild-type allele AGATCT TCTAGA Restriction site Mutant allele AGAGCT TCTCGA Not a restriction site
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