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Stages of Genetic Engineering

DNA cleavage (stage 1) - restriction endonuclease cleaves DNA into fragments 

  • produces large number of different fragments
  • different endonucleases >> different fragments
  • gel electrophoresis - procedure that separates fragments based on size

recombinant DNA production (stage 2) - DNA fragments inserted into vectors 

  • vectors cleaved w/ same restriction endonuclease as DNA

cloning (stage 3) - more recombinant DNA created 

  • vectors introduced into reproducing cells
  • each reproduced cell contains recombinant DNA

screening (stage 4) - most challenging part of any genetics experiment 

  • 4-I - preliminary screening of clones
    • gets rid of cells w/o any vectors, vectors w/o original DNA
    • uses vector w/ gene for antibiotic resistance
    • based on presence/absence of a certain phenotype
  • 4-II - finding gene of interest
    • hybridization - uses complementary nucleic acid (probe) to find particular fragment
    • solution added to denature DNA, allowing radioactive probe to attach

polymerase chain reaction (PCR) - uses DNA polymerase to mass produce gene sequences 

  • denaturation (step 1) - excess of primer mixed w/ DNA fragment
    • DNA strand heated to 98° C >> strands dissociate
  • annealing of primers (step 2) - DNA solution allowed to cool to 60 C
    • DNA strands reassociate w/ excess of primer instead of other complete complementary strand
    • leaves large parts of DNA single-stranded
  • primer extension (step 3) - uses Taq polymerase (heat-stable DNA polymerase) to copy rest of fragment
    • supply of all 4 nucleotides added to solution
    • creates double the amount of DNA as before (separate strands both replicated)
  • cycle repeated to double amount of DNA each time
  • can create large amount of DNA for study from just a tiny amount of DNA

southern blotting - identifies DNA w/ radioactivity 

  • DNA fragmented by endonucleases, spread apart by gel electrophoresis
  • DNA strands denatured by basic gel solution, transferred to nitrocellulose sheet
  • probe w/ single-stranded DNA complementary to gene of interest poured over sheet, will bind to particular sequence

restriction fragment length polymorphism (RFLP) 

  • identifies particular individual w/ specific gene marker
  • mutations, sequence repetitions, transposons alters length of DNA fragments created by endonucleases
  • pattern of bands produced by gel electrophoresis different for each person
  • DNA fingerprinting - 2 individuals rarely produced identical RFLP analyses
    • used in criminal investigations by forensic teams
    • autoradiographs - parallel bars on X-ray film used for comparison
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