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Lecture 16

biology Lecture 16 notes

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Biology 1202B
Brenda Murphy

Lecture 16: Chapter 15—DNA technologies Biotechnology—technology applied to biological systems or living organisms to make or modify products or processes for a specific purpose  Genetic engineering—manipulation of genetic material Bacterial restriction enzymes/Restriction endonucleases (RE)  Discovered in the late 1960’s  RE recognize short amount of DNA sequence (4-8nt long) called restriction sites and cut the DNA at specific locations within those sequences with a restriction enzyme to produce restriction fragments  Restriction sites are often palindromes—same sequence 5’ to 3’ from both strands and produces sticky ends o Ex. 5’ GATC 3’ and 3’ CTAG 5’  Sticky end—can form hydrogen bonds with complimentary sticky ends on any other DNA molecules cut with the same enzyme  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  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 o Conditions include things like temperature, amount of time etc..  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) this happens when a DNA fragment is inserted into a bacterial plasmid  Each human cell has two copies of most genes. On its own this is a very small amount compared to the whole genome, so it is very difficult to study genes. Researchers began cloning fragments so they could amplify them and study them  Clones—a line of genetically identical cells or individuals derived from a single ancestor  Bacterial plasmid: naturally occurring in bacteria, circular, replicate independently from bacterial chromosomes 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 which will be fused with ligase)  This creates some recombinant DNA some nonrecombinant, which is inserted into a bacterial cell  The bacterial grow and divide (replication and amplifies the gDNA)  Identify the bacteria with the gDNA of interest and grow it to produce more  NOTE: the majority of bacterial cells do not take up a plasmid (low transformation efficiency) and plasmids are modified into a cloning vector o i.e. a DNA fragment can be inserted into a plasmid to form a recombinant DNA molecule, which in turn can be amplified in a bacteria  Modification 1: Selection o Insertion of genes to identify which bacteria have a modified plasmid o Give plasmids ampicillin resistance gene, so it can live in ampicillin o Bacterial cells with a modified plasmid will grow
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