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Molecular Genetics and Microbiology
William Navarre

Lecture 14- Transcript MGY377 (Oct.15 recording) NOTE: Slides that were skipped means that the prof just read off of them and didn’t add anything new. They should still be considered testable. Quiz Question #1 Which of the following are characteristics of bacterial spores? A) Have a hydrated cytoplasm that infers heat resistance B) Initiation of spore formation is induced by DNA damage C) Increased PG cross linkages between the spore cortex prevents dehydration D) Spores can actively replicate to produce more spores ANS. A. Cytoplasm with spores have 1/3 of the water content than vegetative cells and that confers heat resitance. C is incorrect as there are decreased PG cross linkages in the spore Quiz Question #2 Choose the incorrect statement regarding antimicrobial peptide: A) Can be produced by bacteria B) Can alter the host immune response C) Sole bacterial target of anti-microbial peptides is the cell membrane D) Anti-microbial peptides are composed of various elements such as alpha- helices and Beta-sheets ANS. C. Antimicrobial peptides can target various cell processes such as cell wall synthesis for instance Microbial Genomics 1 -How it is that we actually sequence a bacterial genome Three major revolutions of microbiology: The germ theory of disease, antibiotics were discovered (anti-microbial resistance), genomics -there are 10x more microbial cells than our own cells Slide 2 -Genomics: branch of study that studies genomes Slide 3 -In order to look at the genome, we need to use sequencing -this technology was first developed in the mid 1970s and was rapidly applied to sequence the first bacteriophage (contained an RNA genome) -took about 20 years until we could sequence our first cellular genome Slide 4-5 -When you compare multiple related genomes is when the real biological significance can be attained -Sanger method: revolutionary method (30 years old) and still used today Slide 6-7 Sequencing using the Sanger method: 1. What you need is a DNA primer and a a piece of DNA. Then you need a DNA primer that is somewhere complementary to that sequence. We need some knowledge of the sequence that we are sequencing (limitation). People get around that by cloning it to a plasmid (and they’ll know the primer sequence). 2. Then you need DNA polymerase and a mixture of nucleotides and you run this in four separate reaction tubes. Each reaction tube contains a nucleotide that will terminate the synthesis of DNA (recall the dideoxy method for sequencing) which includes a dideoxy nucleotide (which misses both of the hydroxyl groups) 3. Extend primer using the DNA polymerase. In this tube, where you dumped it with da, dg, dc, dt and trace amounts of ddc (four nucleotides and one that is defective) it will sequence along and extend a primer. Some random interval along, it has to incorporate a c, it’ll stop (by accidently grabbing a dideoxy c residue) at some percentage of time. You get these staggered fragments. You have millions of polymerases running over millions of fragments in your test tube. 4. Then you run these on a gel (high resolution polyacrylamide gel) the samples move on the basis of size on the four different lanes. Separate basis on the side and you can read back the sequence of the fragments. -Nowadays, we don’t do the reaction in four separate reaction tubes (which contain these fluorescently marked dNTPs) we do it in one reaction tube. We put in four different dideoxynucleotides where each one carries a different fluorescent label I.e. ddT may be yellow and ddA may be blue. Then you run this through a capillary array Slide 10 Now they have machines to do this -Accuracy of this method is very high and the reads are quite perfect. You can only get 1000 bps of reads before it levels out. -Problem is that you don’t know the sequence you need to sequence and you need that primer. Say for instance you have this huge piece of DNA and you don’t know anything about it, how do you predict its sequence? -Take the whole genome let’s say we’re looking at E. Coli genome. Take the chromosomal DNA and shear it up into a bunch of fragments. Randomly throw those sequences into plasmids (now that you have your fragment DNA cloned into a plasmid) and then we sequence left and right ends into our fragments. You do this over and over again and you get reads that overlap with one another. Then you start stitching the genome back together computationally using the reads you get. This will require no knowledge of the sequence. In order to do that, you ned to keep track of which plasmid you have. These plasmid banks = plasmid libraries. You need to have a million different E. Coli plasmids. This is an effort to sequence these plasmids. After you sequence all these plasmids however, you can stitch these together and come up with the sequence. Slide 11-12 Recap of the shotgun-cloning method Slide 13 -Cloning bias: When you cut up some of the genes from S. aureus and put it in E. Coli, some of the genes are toxic. As a result, some of the sequences just won’t clone and now you have a gap in the sequence -you have to maintain a lot of plasmids -its also slow and expensive (took several machines) Slide 14 -Next generation sequencing methods: Pyrosequencing *You need to know this -Sequencing in real time -As you run sequencing reactions (as nucleotides are incorporated), it releases light from the reaction tube when the proper nucleotide is incorporated. You measure sequencing by the bursts of light that is seen. involves a very sensitive camera -Can be preformed with DNA in liquid or with DNA substr
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