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 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 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 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 exact copy of a natural gene Gene can be an altered version of a natural gene Gene can be an altered version of a natural gene Recombinant DNA technology makes it possible 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 Vector: an autonomously replicating genetic element used to carry DNA fragments into a host, typically E. coli, for the purpose of gene cloning Plasmid vectorPlasmid vector Bacteriophage gamma vectorBacteriophage gamma vector Recombinant DNA: any DNA molecule composed of sequences derived from different sources Recombinant DNA: any DNA molecule composed of sequences derived from different sources

5. Cleavage It is done by Restriction enzymes It is done by Restriction enzymes 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 DNA fragments are inserted into vector DNA with the aid of DNA ligases Ligases catalyze the end-to-end joining of DNA fragments Ligases catalyze the end-to-end joining of DNA fragments

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

8. 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 Restriction Enzyme

9. 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 Nuclease

10. Endonuclease TypeCharacteristics I Have both restriction and modification activity Have both restriction and modification activity Cut at sites 1000 nucleotides or more away from recognition site Cut at sites 1000 nucleotides or more away from recognition site ATP is required ATP is required II It has only restriction site activity It has only restriction site activity Its cut is predictable and consistent manner at a site within or adjacent to restriction site Its cut is predictable and consistent manner at a site within or adjacent to restriction site It require only magnesium ion as cofactor It require only magnesium ion as cofactor III Have both restriction and modification activity Have both restriction and modification activity Cut at sites closed to recognition site Cut at sites closed to recognition site ATP is required ATP is required

11. 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 Restriction Enzyme

12. 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 Nomenclature

13. EcoRI E : Genus Escherichia co: Species coli R : Strain RY13 I : First endonuclease isolated BamHI B : Genus Bacillus am: species amyloliquefaciens H : Strain H I : First endonuclease isolated HindIII H : Genus Haemophilus in : species influenzae d : strain Rd III : Third endonuclease isolated

14. Specificity EnzymeSourceSequenceEnd BamHI Bacillus amyloliquefaciens H G  GATCC Sticky BglII Bacillus globigii A  GATCT Sticky EcoRI Escherichia coli RY13 G  AATTC Sticky EcoRII Escherichia coli R245  CCTGG Sticky HaeIII Haemophilus aegyptius GG  CC Blunt HindII Haemophilus influenzae Rd GTPy  PuAC Blunt HindIII Haemophilus influenzae Rd A  AGCTT Sticky HpaII Haemophilus parainfluenzae C  CGG Sticky NotI Nocardia otitidis-caviarum GC  GGCCGC Sticky PstI Providencia stuartii 164 CTGCA  G Sticky

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

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

17. Restriction enzymes

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

21. A typical plasmid vector with a polylinker

22. 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 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" Ends of the plasmid/fragment are closed to form a "recombinant plasmid" Plasmid can replicate when placed in a suitable bacterial host Plasmid can replicate when placed in a suitable bacterial host

25. 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 This can be done by making two cleavages with two different restriction enzymes Construct foreign DNA with same two restriction enzymes Construct foreign DNA with same two restriction enzymes Foreign DNA can only be inserted in one direction Foreign DNA can only be inserted in one direction