Part III. Recombinant DNA technology • Cloning strategies...
Transcript of Part III. Recombinant DNA technology • Cloning strategies...
Chapter 5Part III. Recombinant DNA technology
• Cloning strategies• Polymerase chain reaction (PCR)• Applications
Recombinant DNA technology
• Characterization of the genes• Large production of proteins• Mutants
(Gene cloning, molecular cloning, genetic engineering)
Methodology for transferring genetic information (genes) from one organism to another
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
Polylinker(Multiple Cloning
Site)
Replication origin
ampR
lacZα
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
Tsien at UCSD
GFP
Southern blotDetection 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 polymerasedNTPs
268,435,45630
8,19215
262,14420
8,388,60825
1,073,741,82432
25610
165
44
23
02
01
Number of the target DNA molecules
Cycle number
Exponential amplification
After 4 cycles
After 32 cycles
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
GloFish Giant mouse Dolly, the cloned sheep
Insect-resistant cotton Golden rice
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