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Chapter 16

BY 108 Chapter Notes - Chapter 16: Restriction Enzyme, Molecular Cloning, Southern Blot


Department
BY-Biology Courses
Course Code
BY 108
Professor
Charles Amser
Chapter
16

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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
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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
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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
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