BL5602-L2 Vector.pdf

How to generate transgenic or genetically modified organism? General process 1. Isolation/amplification of selected genes 2. Fusion of selected gene with selected...

How to generate transgenic or genetically modified organism? General process 1. Isolation/amplification of selected genes 2. Fusion of selected gene with selected vector (recombine DNA) 3. Delivery of recombinant DNA into a host cell. 4. Selection of genetically modified host 5. Breeding for offspring with desired genetic modification DNA cloning Recombinant protein production, desirable phenotype How to generate recombinant DNA? 1. Isolation/amplification of selected genes Theoretically, any DNA from any organism can be cloned. DNA can be from genomic DNA, from mRNA, or synthesized. The DNA is cloned for : - (full or partial) protein expression - sequencing - functional studies of the DNA, or its RNA/protein product How to generate recombinant DNA? 1. Isolation/amplification of selected genes Human genome ~3.2 x 109 bp. Average size of a human protein- coding gene (including introns) is ~62,000 bp, ~0.002% of the genome. Human insulin gene is 1,425 bp. How to generate recombinant DNA? 1. Isolation/amplification of selected genes Polymerase Chain denature Reaction anneal extension How to generate recombinant DNA? 1. Isolation/amplification of selected genes Polymerase Chain denature Reaction anneal extension How to generate recombinant DNA? 1. Isolation/amplification of selected genes RE sequence Polymerase Use PCR to introduce new RE sites for cloning. Chain Reaction Primer F Primer R PCR cycles RE site RE site 1. Isolation/amplification of selected genes Polymerase Use PCR to introduce point mutation in the target sequence. Chain point mutation Reaction Primer F Primer R PCR cycles RE site RE site How to introduce point mutation in the middle of the target sequence ? How to generate transgenic or genetically modified organism? General process 1. Isolation/amplification of selected genes 2. Fusion of selected gene with selected vector (recombine DNA) 3. Delivery of recombinant DNA into a host cell. 4. Selection of genetically modified host 5. Breeding for offspring with desired genetic modification DNA cloning Recombinant protein production, desirable phenotype Why need vectors? Vectors are molecular carriers (a DNA molecule too) that carry foreign DNA into another cell. A vector can often replicate itself in the host cell. A vector can be designed to allow transcription and translation of the foreign DNA. A vector can carry antibiotic resistance to facilitate selection of host cells with successful DNA delivery. Common vectors Plasmids Viral vectors (e.g. phage) Cosmids Artificial chromosomes Plasmids – the most commonly used cloning vector 1) Naturally occurred and stably inherited extrachromosomal DNA in bacteria, usually 1-250 kb. 2) Presented as closed circular double-stranded DNA 3) Carrying one or more genes responsible for a useful phenotype, e.g. antibiotics resistance 4) Copy Number is dependent on the form of replication (a) stringent replication: both replication and segregation are under the same control as that of the bacterial genome - 1 to 2 copies per cell. (b) relaxed replication: uncoupled from bacterial genome - many copies per cell (>50 copies) Plasmid vector pBR322 pBR322 is one of the earliest plasmid for DNA cloning (Boyer 1977). pro p- plasmid or mo ot to r An origin of replication for om pr plasmid DNA replication A restrictor of plasmid copy number gene (rop), ~20 copies/cell. Two antibiotic resistant genes (tetracycline and ampicillin, each driven by a promoter) for easy rop selection after foreign DNA insertion. Plasmid vector pBR322 No insert Inserted at BamHI site rop Including both intact and inserted plasmids To identify colonies with intact plasmid Improved plasmid vector Lac promoter/operator (2.7 kb) rop x x White colon Blue colon y Derived from pBR322. y (isopropyl-β-D-1-thiogalactopyranoside) 500-700 copies/cell after removal of rop. pUC plasmids contain a multiple cloning site for easy insertion of DNA IPTG-inducible lac promoter Selection of recombinant plasmids through a reporter gene lacZ for Repressor that is removed by IPTG blue/white colonies Increased cloning capacity Improved plasmid vector Lac promoter/operator (2.7 kb) x x White colon Blue colon y y (isopropyl-β-D-1-thiogalactopyranoside) Repressor that is removed by IPTG pBR322 is the backbone for pUC series and many shuttle vectors