Chapter 19_lecture_Part 1

Genetics: A Conceptual Approach

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Unformatted text preview: Chapter 19- Part I Molecular Genetic Analysis and Biotechnology Dr. Ed Otto George Mason University Key objectives Understand key methodologies used to analyze gene structure and function Briefly discuss some of the applications of recombinant DNA techniques in biotechnology Recombinant DNA Technology Recombinant DNA technology is a set of molecular techniques for locating, isolating, altering, and studying DNA segments The term recombinant is used because the goal is frequently to combine DNA from different sources Recombinant DNA technology = genetic engineering First organisms with recombinant DNA molecules produced by Cohen and Boyer in 1973. Recombinant DNA Technology Recombinant DNA technology has revolutionized the study of genetics A complete industry– biotechnology– has grown up around the use of these techniques to develop new products Working at the Molecular Level Challenges for working with genes at the molecular level: 1.A gene of interest is often just a tiny fraction of the entire genome - a 3000 bp gene constitutes only one-millionth of the human genome (needle in a haystack) 1.If successful in finding gene, must be able to replicate it to make enough copies for study- must be able to successfully amplify DNA in bacteria or in other cells that can be grown in the laboratory 1.Methods to isolate and transfer genes are inefficient- of a million cells exposed DNA, only one cell might successfully take up the gene A. Molecular Techniques Used to Isolate, Recombine, and Amplify Genes- Cutting & Joining DNA Fragments- Viewing DNA Fragments- Restriction Mapping- Southern Blotting- Cloning Genes- PCR Cutting and Joining DNA Fragments Restriction enzymes (or restriction endonucleases) make double-stranded cuts in DNA at specific nucleotide sequences These enzymes are produced naturally by bacteria, where they are used as a defense against viruses - a bacterium protects its own DNA from cleavage by adding methyl groups to its DNA- invading viral DNA is unmethylated and subject to cleavage Enzymes are named for the bacteria from which they are isolated- Eco R1 comes from E. coli; Pvu II from Proteus vulgaris Three types of restriction enzymes have been isolated from bacteria:- Types I and III cut DNA outside of the recognition sequence- Type II restriction enzymes cut DNA at the recognition sequence Virtually all genetic work done uses Type II enzymes More than 800 different restriction enzymes, cutting at more than 100 different sequences, have been isolated from bacteria; many are commercially available Cutting and Joining DNA Fragments Type II restriction enzymes recognize sequences that are usually 4 to 8 bp long Recognition sequences are palindromic : they read the same forward and backward Hin dIII 5’-AAGCTT-3’ 3’-TTCGAA-5’ The longer the recognition sequence, the less frequently it appears in the genome P (six bp) = ¼ X ¼ X ¼ X ¼ X ¼ X ¼ = 1/4096 bp...
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Chapter 19_lecture_Part 1 - Chapter 19 Part I Molecular...

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