RECOMBINANT DNA TECHNOLOGY AND ITS APPLICATIONS SCH2226 HUMAN MOLECULAR GENETICS LECTURE 6: CHAPTER 8 DNA fragments separated by gel electrophoresis and visualized under UV light Student Media Activities iActivity Building a Better Beer —Discover how genetically modified yeasts can improve your brew Animations *DNA Cloning in a Plasmid Vector Restriction Mapping *Dideoxy DNA Sequencing *Polymerase Chain Reaction (PCR) PART 1 RECOMBINANT DNA CHAPTER 8 Restriction site in DNA showing symmetry of the sequence around the center point Restriction site for EcoRI 2 The sequence is a palindrome reading the same from left to right 5**’-to-3**’ on the top strand (GAATTC here) as it does from right to left 5**’-to-3**’ on the bottom strand MUCH LIKE TABLES IN OTHER TEXTS e.g. CUMMINGS . . . . . . BUT THIS ONE HAS A PRONOUNCIATION GUIDE Where do palindromes come from? They arise by chance in both coding and non-coding DNA 3 SmaI produces blunt ends Bam**H1 & Pst**1 produce overhanging (sticky) ends BamHI results in 5**’ overhanging “sticky” ends MORE DETAIL! PstI results in 3**’ overhanging “sticky” ends MORE DETAIL! 4 EcoRI makes staggered, symmetrical cuts in DNA, leaving sticky ends CLONING DNA using restriction A DNA fragment with a sticky end produced by EcoRI digestion can bind by complementary base pairing (anneal) to any other DNA fragment with a sticky end produced by EcoRI cleavage. The gaps are then sealed by DNA ligase CLONING DNA using restriction The plasmid cloning vector pUC19 This plasmid has: an origin of replication (ori) 5 The plasmid cloning vector pUC19 This plasmid has: of the -galactosidase gene lacZ+ The polylinker region is called ‘polylinker region’ BECAUSE it contains multiple cloning sites - many DIFFERENT restriction palindromes into which DNA fragments can be inserted The vector pUC19 contains several unique restriction enzyme sites localized...