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DNA Recombinant Technology

DNA Recombinant Technology. What and Why?. What?: A gene of interest is inserted into another organism, enabling it to be cloned, and thus studied more effectively

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DNA Recombinant Technology

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  1. DNA Recombinant Technology

  2. What and Why? • What?: A gene of interest is inserted into another organism, enabling it to be cloned, and thus studied more effectively • Why?: Detailed studies of the structure and function of a gene at the molecular level require large quantities of the individual gene in pure form

  3. Cloning A collection of molecules or cells, all identical to an original molecule or cell • To "clone a gene" is to make many copies of it - for example, in a population of bacteria • Gene can be an exact copy of a natural gene • Gene can be an altered version of a natural gene • Recombinant DNA technology makes it possible

  4. Terms to Know • Vector: an autonomously replicating genetic element used to carry DNA fragments into a host, typically E. coli, for the purpose of gene cloning • Plasmid vector • Bacteriophage gamma vector • Recombinant DNA: any DNA molecule composed of sequences derived from different sources

  5. Cleavage • It is done by Restriction enzymes • Restriction enzymes : Enzyme produced by bacteria that typically recognize specific 4-8 base pair sequences called restriction sites, and then cleave both DNA strands at this site

  6. Inserting DNA Fragments • DNA fragments are inserted into vector DNA with the aid of DNA ligases • Ligases catalyze the end-to-end joining of DNA fragments

  7. Plasmids Naturally occurring extrachromosomal DNA • Plasmids are circular dsDNA • Plasmids can be cleaved by restriction enzymes, leaving sticky ends • Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid

  8. Restriction Enzyme • Molecular scissors; isolated from bacteria where they are used as Bacterial defense against viruses. • Molecular scalpels to cut DNA in a precise and predictable manner • Members of the class of nucleases

  9. Nuclease Breaking the phosphodiester bonds that link adjacent nucleotides in DNA and RNA molecules • Endonuclease • Cleave nucleic acids at internal position • Exonuclease • Progressively digest from the ends of the nucleic acid molecules

  10. Endonuclease

  11. Restriction Enzyme • There are already more than 1200 type II enzymes isolated from prokaryotic organism • They recognize more than 130 different nucleotide sequence • They scan a DNA molecule, stopping only when it recognizes a specific sequence of nucleotides that are composed of symetrical, palindromic sequence • Palindromic sequence: • The sequence read forward on one DNA strand is identical to the sequence read in the opposite direction on the complementary strand • To Avoid confusion, restriction endonucleases are named according to the following nomenclature

  12. Nomenclature • The first letter is the initial letter of the genus name of the organism from which the enzyme is isolated • The second and third letters are usually the initial letters of the organisms species name. It is written in italic • A fourth letter, if any, indicates a particular strain organism • Originally, roman numerals were meant to indicate the order in which enzymes, isolated from the same organisms and strain, are eluted from a chromatography column. More often, the roman numerals indicate the order of discovery

  13. Nomenclature

  14. Specificity

  15. Restriction enzymes Restriction enzymes can be grouped by: • number of nucleotides recognized (4, 6,8 base-cutters most common) • kind of ends produced (5’ or 3’ overhang (sticky), blunt) • degenerate or specific sequences • whether cleavage occurs within the recognition sequence Become familiar with the back of your molecular biology catalog!

  16. A restriction enzyme (EcoRI) 1. 6-base cutter 2. Specific palindromic sequence (5’GAATTC) 3. Cuts within the recognition sequence (type II enzyme) 4. produces a 5’ overhang (sticky end)

  17. Restriction enzymes

  18. Cloning Vectors Plasmids that can be modified to carry new genes • Plasmids useful as cloning vectors must have • a replicator (origin of replication) • a selectable marker (antibiotic resistance gene) • a cloning site (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers

  19. A typical plasmid vector with a polylinker

  20. Chimeric Plasmids Named for mythological beasts with body parts from several creatures • After cleavage of a plasmid with a restriction enzyme, a foreign DNA fragment can be inserted • Ends of the plasmid/fragment are closed to form a "recombinant plasmid" • Plasmid can replicate when placed in a suitable bacterial host

  21. Directional Cloning Often one desires to insert foreign DNA in a particular orientation • This can be done by making two cleavages with two different restriction enzymes • Construct foreign DNA with same two restriction enzymes • Foreign DNA can only be inserted in one direction

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