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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 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 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 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 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 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 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 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 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 Endonuclease Type Characteristics I II III Have both restriction and modification activity Cut at sites 1000 nucleotides or more away from recognition site ATP is required It has only restriction site activity Its cut is predictable and consistent manner at a site within or adjacent to restriction site It require only magnesium ion as cofactor Have both restriction and modification activity Cut at sites closed to recognition site ATP is required 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 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 Nomenclature EcoRI BamHI HindIII E : Genus Escherichia co: Species coli R : Strain RY13 I : First endonuclease isolated B : Genus Bacillus am: species amyloliquefaciens H : Strain H I : First endonuclease isolated H : Genus Haemophilus in : species influenzae d : strain Rd III : Third endonuclease isolated Specificity Enzyme BamHI BglII EcoRI EcoRII HaeIII HindII HindIII HpaII NotI PstI Source Bacillus amyloliquefaciens H Bacillus globigii Escherichia coli RY13 Escherichia coli R245 Haemophilus aegyptius Haemophilus influenzae Rd Haemophilus influenzae Rd Haemophilus parainfluenzae Nocardia otitidis-caviarum Providencia stuartii 164 Sequence GGATCC AGATCT GAATTC CCTGG GGCC GTPyPuAC AAGCTT CCGG GCGGCCGC CTGCAG End Sticky Sticky Sticky Sticky Blunt Blunt Sticky Sticky Sticky Sticky 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! 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) Restriction enzymes 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 A typical plasmid vector with a polylinker 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 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
