Chapter 5 Part III. Recombinant DNA technology • Cloning strategies • Polymerase chain reaction (PCR) • Applications Recombinant DNA technology (Gene cloning, molecular cloning, genetic engineering) Methodology for transferring genetic information (genes) from one organism to another • Characterization of the genes • Large production of proteins • Mutants Tools of recombinant DNA technology • Restriction endonucleases - cut DNA at specific sites • DNA ligase or other DNA modifying enzymes • Cloning vectors - DNA molecules that can be replicated • Reporter genes • Model organisms Restriction enzymes • Bacteria’s “immune system” for protection from infection by foreign DNA • Three types – Type I and Type III : Both the endonuclease and the methylase activity; Remote recognition site – Type II : Only endonuclease activity; Specific and predictable recognition; • Cohesive (sticky) or blunt ends Palindromic restriction sites Restriction map Restriction map Restriction-fragment length polymorphism (RFLP) Inheritance of RFLPs Cloning vectors • Plasmids – Replication origin, selectable marker & polylinker – Stringent control (low copy number) or relaxed control (medium to high copy number) • Viral vectors – Bacteriophage λ, cosmid & M13 : Bacteria – Baculoviruses: Insects – Retroviruses, lentiviruses & adenoviruses: Mammalian cells • Yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) Plasmid cloning vector lacZα Polylinker (Multiple R amp Cloning Site) Replication origin Construction of recombinant DNA Cloning in λ phages Cloning strategies • DNA ligase •PCR • Terminal transferase • Adaptor • Topoisomerase • Recombinase Cloning using DNA ligase Cloning using terminal transferase Cloning using synthetic adaptor Reporter genes • Selectable markers – Antibiotic resistance – Nutritional markers • LacZ (β-galactosidase) • Luciferase • Green fluorescence protein (GFP) Insertional gene inactivation (Replica plating) β-Galactosidase as a reporter GFP as a reporter GFP Tsien at UCSD Southern blot Detection of specific DNA sequence Colony (in situ) hybridization Identification of the clones containing a DNA of interest Model organisms Polymerase Chain Reaction (PCR) Technique for the exponential amplification of a specific DNA segment • Template DNA • Two oligonucleotide primers • Heat stable DNA polymerase (Taq, Pfu etc) Three step process • Template denaturation • Primer annealing (hybridization) • Primer extension (polymerization) Denaturing Annealing Extension Heat stable DNA polymerase dNTPs Exponential amplification Number of the Cycle target DNA number molecules 1 0 2 0 3 2 4 4 After 4 cycles 5 16 10 256 15 8,192 20 262,144 25 8,388,608 30 268,435,456 After 32 cycles 32 1,073,741,824 Application of PCR • Clinical applications - Diagnosis of infectious diseases and rare mutations • Forensics – DNA fingerprinting • Molecular archeology – Evolutionary study • Asymmetric PCR – DNA sequencing • Site-directed mutagenesis Site-directed mutagenesis Applications of the recombinant DNA technology • Recombinant proteins – Research – Medical purposes • Genetically altered organisms – Transgenic – Knockout • Gene therapy Recombinant proteins Genetically modified organisms (GMOs) • Bacteria – Bioremidation, biomining, biofuel etc • Plants – Resistace to pests, herbicides or harsh environmental conditions; improved shelflife; increased nutritional value • Animals – Transgenic or cloned animals Insect-resistant cotton Golden rice GloFish Giant mouse Dolly, the cloned sheep Approaches for gene therapy • ex vivo – Treatment of cells with a vector outside of the body • in situ – Direct application of the vector to affected tissues • in vivo – Direct injection of the vector into the blood stream .