1362-SP10-Lecture-7-_40962 - DNAresearchinthe50s,...

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DNA research in the 50’s, 60’s progressed with the concurrent Recombinant DNA Technology development of tools for the analysis of DNA. Culminated in the discovery of restriction enzymes & the formation of recombinant DNA molecules in the early 70’s. • Recombinant DNA = combining pieces of DNA from different sources to produce hybrid DNA molecules. (called “cloning DNA”) Generally one recombines DNA fragments to produce hybrids containing Generally, one recombines DNA fragments to produce hybrids containing one or a few genes of interest. Relies on the fact: all DNA is structurally/chemically the same • Recombinant DNA technology serves as the foundation to exploit biological processes for practical applications; i.e., biotechnology . Combines the fields of biology, chemistry, & engineering. Biotechnology process: basic research, trials, scale up Applications in pharmaceuticals, industrial products & processes, agriculture, & textiles • Recombinant DNA technology has also been essential to advancements in understanding many biological process.
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Overview of DNA Cloning 1) Source DNA: isolate DNA from cells containing gene(s) of interest 2) Bacterial plasmid vector : small, circular, extra chromosomal DNA; gene(s) of interest inserted into plasmid into plasmid. 3) Cut DNA & vector with a restriction enzyme & combine t f bi t l l to form recombinant molecules 4) Amplify gene: transform host cells & grow; cell population contains cloned gene. 5) Applications: research on the gene or on the gene product (protein)
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Restriction Enzymes Gene cloning relies on restriction enzymes to cut DNA molecules at a limited number of specific locations. recognize specific sites (4 to 8 bp long) on double stranded DNA Restriction sequences are palindromic; read the same in 5’ to 3’ direction on Restriction sequences are palindromic; read the same in 5 to 3 direction on both strands (both strands are cut ) Restriction enzyme from E. coli bacterium EcoR1 recognizes EcoR1 binds to DNA at these sequences & l i di d bacterium, EcoR1, recognizes DNA sequence: 5’ G*AATTC 3’ 3’ CTTAA*G 5’ 5’ …A T T C A G * A A T T C G C T T A… 3 3’ T A A G T C T T A A * G C G A A T 5’ & cleaves as indicated: 5’ AATTC GCTTA 3’ 3 CTTAA G 5 3 …T A A G T C G A A T… 5 Cleavage by EcoR1 generates restriction fragments with “sticky ends”; ends have an overhang of single stranded DNA 5’ …ATTCA G 3’ 3’ …TAAGT CTTAA 5’ 5 GCTTA… 3 3’ G CGAAT… 5’ DNA. “sticky DNA li ends” Combine with fragment from another DNA cut w/same restriction enzyme DNA ligase seals strands
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Using Restriction Enzymes Cut the following sequence with the restriction enzyme Taq I (recognizes 5’ T*CGA 3’ ): How many fragments? 5’ C T C G A T T A T C G A A C C T T C A T C G A T T C 3’ 5 C T C G A T T A T C G A A C C T T C A T C G A T T C 3 3’ G A G C T A A T A G C T T G G A A G T A G C T A A G 5’
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• Derived from naturally occurring bacterial plasmids.
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