08 Genetic engineering

08 Genetic engineering - Recombinant DNA and genetic...

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Recombinant DNA and genetic engineering
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Principles of recombinant DNA technology Active growth since the mid-1970s Genetic engineering applies recombinant DNA technology to problems in biology, medicine, and agriculture Genomics studies information contained in the genome at the molecular level Recombinant DNA technology depends on 4 basic principles: 1. The ability of single-stranded polynucleotides to base pair with their complement
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The 4 basic principles 3. ability to join DNA molecules, creating recombinant DNA 4. ability to amplify any recombinant DNA to generate large amounts 2. ability of proteins to recognize target DNA sequences
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How do you make recombinant DNA? First, you need a DNA source: genomic DNA from chromosomes. However, this is usually too large to clone directly cDNA (complementary DNA) derived by action of reverse transcriptase from (usually) mRNA template PCR fragments chemically synthesized oligonucleotides
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Restriction enzymes cut the DNA where you chose recognize palindromic double- stranded sequences produce complementary “sticky” ends dozens of such enzymes sticky ends can be ligated under appropriate conditions Digest DNA with a restriction enzyme
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3. Use cloning vectors for mass production cloning vectors permit replication of inserted DNA include plasmids, vectors, artificial chromosomes complementary restriction ends (sticky ends) joined by DNA ligase multiple fragments can be joined Clone the DNA into a vector
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4. Transformation into an expression system (bacterial cell, e.g., E. coli or eukaryotic cell, e.g., yeast) = little factories for making your DNA Amplify the recombinant DNA
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Amplify the recombinant DNA Requires the use of cloning vector or artificial chromosome The recombinant DNA molecule must enter host cell by transformation Use an antibiotic to kill all cells without the recombinant DNA. Only the transformed bacteria are left The recombinant molecule must efficiently replicate with the plasmid DNA inside the transformed bacteria (usually grow the bacteria overnight) Large amounts of the replicated plasmid DNA can be recovered from the transformed bacteria using a plasmid prep kit available form many biotechnology companies
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Cloning in a host cell Requires use of cloning vector or artificial chromosome Recombinant molecule must enter host cell Use antibiotic to kill cells without the recombinant DNA Recombinant molecule must efficiently replicate Replicated plasmid DNA must be recovered from host clone Many commercial products available
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Plasmid cloning vectors – the workhorse Common properties: origin of DNA replication so the plasmid can replicate multiple cloning sites containing many restriction sites engineered into many plasmid vectors drug resistance gene to select for bacteria that took up your plasmid (ampR)
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Bacteriophage lambda DNA to clone Phage vector
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This note was uploaded on 04/23/2008 for the course BY 214 taught by Professor Woodworth during the Spring '07 term at Clarkson University .

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08 Genetic engineering - Recombinant DNA and genetic...

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