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Genetic Engineering - Genetic Engineering WHAT IS GENETIC...

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Genetic Engineering WHAT IS GENETIC ENGINEERING? • This is the term we used to describe the process of manipulating genes outside an organism. DAY TO DAY IMPACT • Production of Pharmacologics – Insulin (1982) – Factor VIII hemophilia • Analysis of Mutations (in utero) • Forensics (CSI) • Heat Stable Proteases in Detergents • Insect Resistant Plants • Gene Therapy • Vaccines (hepatitis B, 1986) USING WHAT YOU KNOW All of the techniques we will discuss will rely on principles we have already studied. Base pairing Replication Transcription Transcription factors and enhancers BRIEF HISTORY LESSON • 1953: Double Helix (Watson & Crick)* • 1961: DNA Renaturation (Marmur & Doty) • 1962: Restriction Enzymes (Arber, Nathans & H. Smith)* • 1966: Cracking of Genetic Code (Nirenberg, Ochoa, & Khorana)* • 1967: DNA Ligase (Gellert) • 1970: Reverse Transcriptase (Temin & Baltimore)* • 1973: Cloning/Plasmids (Cohen, Chang, Helling, & Boyer) • 1977: DNA Sequencing (Sanger, Gillbert & Berg)* • 1986: PCR (Mullis)* • 1999: “Dolly” • 1995-2000: DNA Arrays, Sequencing of Human Genome, Yeast, C. elegans INITIAL PROBLEMS: How do you get a specific gene out of the chromosome? A gene IS NOT an isolated entity like a protein
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If cells part of compact tissue, dissociate using proteolytic enzymes and other agents to disrupt adhesive bonds, tissue then teased apart in single cells INITIAL PROBLEMS: How do you get Expression of a specific gene Recall: Introns and Exons Gene doesn’t exist as single entity. Cut (cleave) double stranded DNA at certain sites determined by seq nuc pairs using restriction nucleases. Produce reproducible set of specific DNA frags. INITIAL PROBLEMS: How do you get DNA into a specific cell? Cells have membranes! Need to get into the correct cell INITIAL PROBLEMS: How do you determine what controls the expression of a specific gene? WHAT WE NEED We need to be able to isolate or manipulate: genes (exons and introns) DNA sequences of interest (promoters, enhancers, etc) mRNA (complete coding sequences) Protein (functional proteins such as tfs or enzymes) MANIPULATION OF DNA CRITICAL DISCOVERY: Bacteria express specific enzymes that physically “cut” (break) the DNA at specific sites (4-8bp) RESTRICTION ENDONUCLEASES (aka: Restriction Enzymes) DISCOVERY DNA injected into a bacteria was degraded. Thus, enzymes in the bacteria “resticted” the transfer of the exogenous DNA KEY CONCEPT Each bacteria expresses a different restriction enzyme and these recognize a different sequence of DNA KEY CONCEPT Because each restriction enzyme (RE) has a different specificity, and cuts a sequence that is usually 4-8bp, a given RE sequence will occur in nearly any stretch of DNA. Can be used to analyze DNA from any source 4bp sequence will occur every 256bp (44) 6bp = 1:4096 (46)
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8bp = 1:65,000 (48) PALINDROMES • Most RE recognize a sequence that is called a palindrome.
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