Biology 1202B Lecture Notes - Dominance (Genetics), Galactose, Mutagen

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30 Apr 2012
Department
Biology Lecture Review Notes
Lecture 6
DNA Technologies
Biotechnology - Technology applied to biological systems or living organisms to make or modify
products or processes for a specific purpose
o Genetic engineering manipulation of genetic material
Bacterial Restriction Enzymes or Restriction Endonucleases (RE)
o RE recognize short amount of DNA sequence (4-8nt long) called restriction sites
o Restriction sites are often palindromes - same sequence 5’ to 3’ on both strands
Example: 5’GATC 3’ and 3’CTAG 5’
o RE cut DNA within these restriction sites
o Complete digestion - conditions are such that if a restriction enzyme site is present in
the dsDNA, the enzyme will cut at all the dsDNA sites
o Incomplete digestion - conditions are such that if a restriction enzyme site is present in
the dsDNA, the enzyme may cut at all the dsDNA sites
Conditions include things like temperature, amount of time, etc.
o Sticky end can bind back together after being cut by a RE
o Another DNA fragment (complementary to the strands) from EcoRI digestion can bind to
these sticky ends, but need DNA ligase to bind it all together (seal the nicks)
o Blunt cutter cuts straight through DNA and does not create a sticky end
Clones a line of genetically identical cells or individuals derived from a single ancestor
o Cloning older method of amplifying cells and gaining identical daughter cells for
further studying
o Cloning a DNA fragment into a bacterial plasmid
Isolate gDNA and RE cuts it into fragments
The same RE cuts a bacterial plasmid
Mix the cut gDNA with the cut plasmid (they have the same sticky ends)
This creates recombinant DNA, which is inserted into a bacterial cell
The bacteria grow and divide (replicates and amplifies the gDNA)
Identify the bacteria with the gDNA of interest and grow it to produce more
o Low transformation efficiency the majority of bacterial cells do not take up a plasmid
o Plasmids are modified into a cloning vector (something that can accept foreign DNA)
o How to determine which bacterial cells have modified plasmids
Selection - insertion of genes to identify which bacteria have a modified plasmid
Give plasmids ampicillin resistance gene, so it can live in ampicillin
Bacterial cells with a modified plasmid will grow in ampicillin
Bacterial cells without a modified plasmid will not grow in ampicillin
This will eliminate all the plasmids without fragments
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