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The DNA Toolbox
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The Human Genome Project
Genome- all of an organism’s DNA Mapped 3.2 billion base pairs Between 20,000 and 30,00 genes Completed in 2003 A = T C = G
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Biotechnology Manipulation of organisms or their components to make useful products Historically, it includes selective breeding of farm animals and crops, and using microorganisms to make wine and cheese Today, it also includes genetic engineering
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Genetic Engineering Direct manipulation of the DNA molecule itself
Utilizes recombinant DNA
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Recombinant DNA DNA molecules formed when segments of DNA from two different sources (often different species) are combined in vitro (in a test tube)
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Genetic engineering requires a special set of tools
This unit is a survey of those tools…
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Review Genes occupy only a small proportion of a chromosome
The coding sequences are called exons The rest of the chromosome is made up of non-coding segments called introns exon intron gene
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DNA Cloning Scientists needed a way to work directly with desired genes and not the “trash” DNA cloning is the process by which multiple identical copies of specific DNA is produced
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E coli Most cloning methods involve bacteria
Most commonly used bacteria is E coli
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Bacteria Review Unicellular prokaryotes Reproduce by binary fission
Short generation span New offspring every +/- 20 minutes Colony of 100 million overnight! Incredibly diverse Dominant form of life on Earth
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Bacterial Genome Single, circular chromosome
Reproduce asexually so always haploid (n) Naked DNA (no histones) ~4 million base pairs; 4300 genes 1/1000 DNA in eukaryote Contain plasmids Genome = all the DNA of an organism Eukaryotes • 1000 times more DNA • only 10 times more genes - introns, spacers, inefficiency
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Plasmids Small supplemental circles of DNA
Replicate separately from bacterial chromosome Carry 2-30 genes, including those for antibiotic resistance Can be imported from environment
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Inserting Genes into Bacteria Using Plasmids
Cut plasmid DNA and DNA containing desired gene Insert new gene into plasmid Insert recombined plasmid into bacteria transformed bacteria gene from other organism recombinant plasmid vector plasmid cut DNA + glue DNA
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How do we cut DNA? Restriction enzymes
Evolved in bacteria to cut up foreign DNA Protect bacteria against invaders Bacteriophages- viruses that attack bacteria
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What keeps the restriction enzyme from cutting up the bacteria’s own DNA?
Methylation By using enzymes that DO NOT recognize any of their own base sequences
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What do you notice about these phrases?
radar racecar Madam I’m Adam a man, a plan, a canal, Panama Was it a bar or a bat I saw? palindromes
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Restriction Enzymes Target Palindromes
Which of the following DNA sequences is a palindrome? A B C. 5' AAGG 3' 5' AGTC 3' 5' GGCC 3' 3' TTCC 5' 3' TCAG 5' 3' CCGG 5'
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Inserting Foreign DNA into a Plasmid
Restriction enzyme reads DNA from 5' to 3' looking for specific sequence: GAATTC Cuts DNA at restriction site: between G and A Most useful produce asymetrical cuts resulting in protuding ends called sticky ends Sticky ends will bind to any complementary DNA CTG GACTTAA 5' 3' AATTCCG GGC CTGAATTCCG GACTTAAGGC
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Many Different Restriction Enzymes
Discovered in 1960s Key to genetic engineering Named after organism in which they were found E coli Restriction Enzyme I (EcoRI): first to be discovered in E coli Each one recognizes a unique base sequence so they cut in different places
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Common Restriction Enzymes
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chromosome want to add gene to
Sticky ends Cut other DNA with same enzymes leave “sticky ends” on both can glue DNA together at “sticky ends” DNA Ligase- enzyme that glues sticky ends together GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA gene you want GGACCTG AATTCCGGATA CCTGGACTTAA GGCCTAT chromosome want to add gene to GGACCTG AATTCACGCTT CCTGGACTTAA GTGCGAA combined DNA
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The code is universal Since all living organisms… use the same DNA
use the same code book read their genes the same way Strong evidence for a single origin in evolutionary theory.
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Plasmids and Genetic Engineering
cut DNA using restriction enzyme gene we want like what? …insulin …HGH cut plasmid DNA using same enzyme recombinant plasmid capable of producing desired protein insert “gene we want” into plasmid... “glue” together using DNA Ligase
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Transformation Insert recombined plasmid to form transformed bacteria
Grow bacteria allowing it to produce protein as directed by new gene Harvest & purify protein
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ANY QUESTIONS?
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