1 / 26

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

Télécharger la présentation

DNA Recombinant Technology

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.


Presentation Transcript

  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

More Related