Molecular Biotechnology-Lession 2: Recombinant DNA technology docx

Recombinant DNA technology (Gene cloning, genetic engineering) VIETNAM NATIONAL UNIVERSITY AT HO CHI MINH CITY INTERNATIONAL UNIVERSITY Tran Ngoc Duc, PhD 2. Prokaryotic and eukaryotic genes 3. Vectors 4. Cloning a gene 5. Transformation technology 6. Detection of the cloned gene/products 1. Introduction 1/6 Introduction  Glofish is the first transgenic animal approved to be consumed by human in the USA  The insertion of different constructs of GFP into the fish genomes to give different green colors [...]... bacteria  Can carry up to 25kbDNA  1000 time more efficient than a plasmid Cloning in Lambda Vectors 1 Make a partial digest of DNA 2 Ligate the DNA to the arms of the vector 3 Package the DNA into phage particles using premade mixes 4 Screen and then amplify 5 Store the library for future use as a plate lysate  Cosmid o A combination of a plasmid and a cos site (DNA sequence, 200bp originally... at each chromosome end, protects the linear DNA from degradation by nucleases  CEN: The centromere which is the attachment site for mitotic spindle fibers, "pulls" one copy of each duplicated chromosome into each new daughter cell  ORI: Replication origin sequences which are specific DNA sequences that allow the DNA replication machinery to assemble on the DNA and move at the replication forks 4/6... forks 4/6 Cloning a gene http://www.ndsu.edu/pubweb/~mcclean/plsc731/cloning/cloning2.htm  Cloning a gene Gene /DNA isolation Ligation gene /DNA to a vector Transform ligated vectore into cell/tissue/ organism Screening the transfered gene 5/6 Transformations technology  Transformations technology  Heatshock  Electroporation  Injection  Bombarment/gene gun  Mediation of Agrobacteria  Eukaryote... beta-lactamase  Origin of replication, multi-cloning site and selectable marker are common to vectors  Self-replicating  Double stranded  Circular DNA  Independent extrachromosomal DNA in  Ampicillin bacteria 1kb-500kb  Kanamycin  Origin of DNA replication, ori  High (10-100) & low copy plasmid (1-4)  Neomycin Tetracycline  Streptomycin  Hygromycin  White blue screening  LacZ encode... essential for packaging) which help to transfer a plasmid into a phage head o Can carry up to 52 kb DNA o Unlike plasmid, cosmid can also packaged into a capsid since it contains the cos site and so it can  Yeast artificial chromosome (YAC): o To clone large piece of DNA which can carry up to 2-3 MbDNA o Constructed from the telomeric, centromeric, and replication origin sequences needed for replication... plant, either in root or trunk  The tumor is caused by a plasmid called tumorinducing plasmid (Ti-plasmid or Ri-plasmid)  Specifically, the tumor is caused by a conjugative DNA fragment on the plasmid, which is called T -DNA  T -DNA carries genes for auxin, cytokinin, nopaline  Altering balance of plant hormone => cell division is not controlled ...  Heatshock with E coli  Making competent cells with ice-cold CaCl2  A mixture of E coli and vector  Expose to 2 min at 420C  Frequency: 1 out of 1000  Efficiency: 107- 108 colonies/µg of plasmid DNA  Electroporation  Subject bacteria to high voltage electric field  Efficiency: 109 colonies/µg of small plasmid (3kb), 106 colonies/µg of large plasmid (136kb)  Mediated by Agrobacteria  Agrobacteria:... Eukaryotic  Eukaryotic genes are more complex than prokaryotic genes  Intron: noncoding region, 40-10,000bp  Exon: coding region, 150-500bp  5’, 3’ UTR = flanking regions 3/6 Vectors  Vector is a DNA molecule used as vehicle to transfer and amplify a foreign genetic material into another cell  There are four types of vector: o Plasmids o Virus o Cosmids o Artificial chromosomes (BAC, YAC)  Depending . Company  (http://en.wikipedia.org/wiki /Recombinant_ DNA)  Applications of recombinant biotechnology  Recombinant human insulin  Recombinant human growth hormone  Recombinant human. using recombinant DNA technology was human insulin, developed by Genentech and Licensed by Eli Lilly and Company  (http://en.wikipedia.org/wiki /Recombinant_ DNA)  Applications