Lecture 24, 11/7 (Dr. Maybruck 9); Bacterial Genetics

Audio for today's lecture is available here.

• • Slide 1 (handout from 11/5) tools and techniques for biotechnology applications: Plate hybridization
• Plate hybridization - Helps identify specific DNA sequence associated with an organism
• You use plate hybridization in order to determine if an organism is present and is harmful by looking at the DNA.
• Take your food sample and streak a plate –-> incubate plate for a day → bacteria grow. From this growth we do gene analysis.
• Place nitrocellulose on top of the colonies (so that they touch) that have grown. Pull the membrane off and it contains smears of the colonies that had grown on the plate. Next you lyse the cells and denature the DNA. Denaturing the DNA causes it to separate into single strands. You then expose the membrane to a gene probe. The gene probe is a sequence of DNA that will complimentary bind to the gene of interest (the one that has the toxins). If there is no harmful gene then the gene probe will not bind. The gene probe has a radioactive label that emits energy. The photographic film picks up the radioactive label (if it is bound to the DNA in one of the colonies) and a mark appears on the photographic film. You can then take the photographic film, lay it over the original colony again, and determine which colonies have the genes of interest.
• • Slide 2 – PCR
• Polymerase chain reaction allows us to amplify specific sequences of DNA.
• Components of a test tube:
• First thing you need is your DNA fragment that you wish to amplify. Then you need DNA polymerase which is responsible for synthesizing the new complimentary strand of DNA (semiconservitive replication). DNA polymerase will not add complimentary bases to make a new strand of DNA without primers. When making a complimentary new strand of DNA you need nucleotides.
• The above components will be put into a thermocycler (instrument that cycles through temperatures).
• 3 steps occurring in one cycle of PCR (the more cycles the more DNA molecules)
• • denaturization – heat can be used to separate the DNA strands to separate at 94ºC
• • priming – temperature is cooled to 50-65ºC. this causes the primers to bind were they need to. This is important because DNA polymerase wont start adding new nucleotides until there is a primer there.
• • Extension – temp is increased to about 70º which causes DNA polymerase to come in and start making new strand of DNA.
• Thermos aquaticus – thermophile. Organism able to survive in extreme heat.
• • Taq polymerase – used often in PCR because its polymerase is used to being at hot temperatures.
• Video explanation titled “Polymerase Chain Reaction” – available on blackboard
• • Slide 3 DNA sequencing
• DNA sequencing: Gives us an idea of what we are dealing with
• Sanger dideoxy method of DNA sequencing
• • Dideoxy nucleotide is missing an oxygen from its 3 carbon sugar.
• In a test tube you have: gene, primers, DNA polymerase, 4 nucleotides (G,C,A,T), 1 modified nucleotide (this is the dideoxy nucleotid missing oxygen from its 3 carbon sugar)
• Once the modified nucleotide is added to the chain then nothing will add on afterwards.
• Video titled “SANGER SEQUENCING” available on blackboard
• • Slide 1 (handout 11/7)
• Recombinant DNA is taking the genetic material of one organism and incorporating it into the DNA of another
• This is a natural occurrence among bacteria. It is called transformation.
• We can use this to our benefit as we can make organisms make proteins, Make transgenic organisms and amplify DNA.