12-Gene Cloning.ppt

Gene Cloning 1 Learning objectives • What is gene clonning ? • Elements of gene clonning. • Steps of successful gene clonning: insertion, transformation and identification. 2 • 1. 2. 3. 4. 5. Gene cloning is the introduction of a foreign DNA molecule into a replicating cell permits the ampl...

Gene Cloning 1 Learning objectives • What is gene clonning ? • Elements of gene clonning. • Steps of successful gene clonning: insertion, transformation and identification. 2 • 1. 2. 3. 4. 5. Gene cloning is the introduction of a foreign DNA molecule into a replicating cell permits the amplification (that is, the production of many copies) of the DNA. A single DNA fragment (a nucleotide sequence of interest ) can be isolated and purified prior to cloning. This involve the following steps: Extract and purify DNA from cells. Cut DNA with restriction enzyme Separate DNA fragments by “Agarose gel electrophoresis” Use of Southern blot technique: a. Transfer DNA from the fragile gel to a nylon sheet and heat to sep. strands b. Hybridize gene of interest with a radio-labeled DNA or mRNA probe and expose w/ film to locate gene. Gene cloning. 3 • Expression Vector: An expression vector, also known as an expression construct, is usually a plasmid designed for protein expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. The plasmid is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription and translation of the gene carried on the expression vector. 4 • Characteristics of Expression vector (Plasmid vector): 1) origin of replication, 2) cloning site for the insertion of a gene, 3) strong promoter, 4) a transcription termination sequence, 5) correct translation initiation sequence such as a ribosomal binding site and start codon, a strong termination codon, 6) Antibiotic resistance sequences 5 Structural features of an expression vector (Plasmid) polylinker 6 1- Insertion 7 2- Transformation 8 Chemical Transformation using Calcium Chloride 9 Transformation by Electroporation 10 3- Identification/ Step I - Grow transformed bacterial cells on medium containing ampicillin. Cells containing plasmids contain the ampicillin resistance gene and survive. Survived cells/clone may contain recombinant or non-recombinant palsmids. - How do you know which colonies contain the recombinant plasmid i.e. gene of interest ? 11 3- Identification/ Step II A) Utilization of radioactive DNA probe 12 B) Blue/White screening: • Only E. coli cells with resistant plasmids grow on antibiotic medium • E. coli cells with plasmids with functional lacZ gene can grow in presence of Xgal substrate (inducer is required – IPTG = Isopropylthiogalactoside). lacZ(+) blue colonies = no insertion lacZ(-) white colonies = polylinker is disrupted = successful insertion. 13 * Gene Cloning (Summary) 14