Genetic engineering 3 

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  • Genetic engineering 3
    • PCR
      • Method used to amplify minute amounts of dna by repeated cycles of in vitro dna replication. Amplification proceeds in exponential scale
      • What do you need? A small amount of dna (template). The exact sequence at the start and end needs to be known to make primers (15-25 in length). DNA Polymerase (taq) and lots of nntp's. Also machine that can hold tubes at different temperatures"thermal cycle"
        • Primer at 5' end (beginning) is derived from sense strand/ The  one at the 3' end is derived from anti-sense strand
      • PCR cycle; 1) denaturation at 94-96 degrees 2) Annealing at 68 3) Elongation at 72
      • What is it used for? 1) Detection - genotyping mutants, infectious agents and forensics 2) Amplification to obtain enough dna for cloning 3) Creating mutations
        • PCR to insert mutations. Possible as primers are incorporated.Primers can add suitable restriction enzyme sites to enable subsequent cloning of the product
    • Cloning on DNA
      • Maintains dna of interest in isolation and outside its biological context. Generates unlimited supply of it.
      • Involves inserting a piece of dna into a host dna molecule known as the vector. The insert can be a whole gene or part of it.
        • The vector = a dna molecule that is maintained and replicated naturally by a host organism (bacteria, virus) Good as short life span, easy and cheap to grow, easy to break up
          • Plasmids are the most common vector. They are small circular DNA (3kbp) found naturally in bacteria
      • Essential properties of a cloning plasmid; contains an origin of dna replication (Ori) to allow it to exist and replicated independently of the bacterial chromosome
        • Contains an antibiotic resistance gene to select for bacteria containing it. Also have unique enzyme restriction sites.
      • Plasmids
        • Types; cloning (e.g. pBluescript), expression plasmids (bacterial, mammalian). An expression vector is a cloning plasmid. The reverse is not true
        • Expression vectors must contain a promoter to initiate transcription
    • Steps in cloning
      • 1. Generation of compatible cohesive ends in insert and plasmid 2) fusion of insert to plasmid using dna ligase
        • 3) Introduction of recombinant dna molecules into bacteria for amplification 4) Antibiotic selection for bacteria containing plasmids 5)Large scale propagation of bacterial colonies
      • DNA libraries; population of identical vectors each containing a different insert. Can be cdna (dna made from mrna), or dna.
        • Two types; one one contains the complete dna sequence of an organism e.g. mouse library and cdna libraries only represent the part of genome that is expressed and can be derived from different organism and stages in development
          • libraries can be kept in solution (Test tube) or plated


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