Lecture 7 - Microbial Genomics.docx

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Department
Cell and Systems Biology
Course Code
CSB328H1
Professor
William Navarre

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MGY377H © Lisa| Page 113 L E C T U R E 7 : M I C R O B I A L G E N O M I C S HOW TO SEQUENCE A MICROBIAL GENOME (FROM 1976 TO 2013) 1. genomics is the branch of science/technology which specializes in the systematic study of: 1. genomes (including their molecular characterization) 2. & the production of their gene product (proteins), 3. their role in health & disease, 4. & the effects of manipulation of these systems by agents such as pharmaceuticals & radiation 2. sequencing: procedures for determining of exact order of nucleotides in the DNA fragment 3. sequencing virus genomes 1. 1976: MS2 phage 2. 1977: Phi-X174 phage (Fred Sanger et al.) 3. these 2 bacteriophage genomes were the first to be determined in history 4. first cellular genome: Haemophilus influenzae (1995) 1. In 1995, Haemophilus influenzae was the first free-living organism to have its entire chromosome sequenced, sneaking in just ahead of Escherichia coli in that race, mainly because its genome is smaller in size than E. coli's. For a relatively obscure bacterium, there was already a good understanding of its genetic processes, especially transformation. 5. steps towards DNA sequencing 1. the plus & minus method (1975) was one of the first methods used to sequence DNA, & required a comparison of both the “plus” & “ minus” sequences to determine the actual sequence 1. it could be used ONLY for sequencing ssDNA 2. the Maxim & Gilbert method (1977) was published just before the inhibitor method, however it required a series of complex rxns & was therefore a much more difficult method of sequencing 1. like the inhibitor method, it can be applied to dsDNA 3. the Sanger et al method (1977) was a method that incorporated the idea that inhibitors can terminate elongation of DNA at specific points 1. this technique remains the standard today THE SANGER METHOD 6. this method is based on the DNA polymerase-dependent synthesis of a complementary DNA strand in the presence of natural 2’ deoxynucleotides (dNTPs) & 2’,3’-dideoxynucleotides (ddNTPs) that serve as non-reversible synthesis terminators 7. the DNA synthesis rxn is randomly terminated whenever a ddNTP is added to the growing oligonucleotide chain, resulting in truncated products of varying lengths w an appropriate ddNTP at their 3’ terminus MGY377H © Lisa| Page 213 8. modern automated DNA sequencing 1. originally, 4 diff rxns were required per template, each rxn containing a diff ddNTP terminator: ddATP, ddCTP, ddTTP, ddGTP 2. advances in fluorescence detection have allowed for combining the 4 terminators into 1 rxn by having them labeled w fluorescent dues of diff colours 3. overall throughput has been increased by the advent of capillary arrays whereby many samples could be analyzed in parallel Automated DNA sequencing utilizes a fluorescent dye to label the nucleotides instead of a radioactive isotope. The fluorescent dye is not an environmentally hazardous chemical and has no special handling or disposal requirements. Instead of using X-ray film to read the sequence, a laser is used to stimulate the fluorescent dye. The fluorescent emissions are collected on a charge coupled device that is able to determine he wavelength. The Perkin-Elmer Applied Biosystems (ABI) DNA sequencers are designed to discriminate all four fluorescent dye wavelengths simultaneously, which allows for complete DNA sequencing in one lane on the gel. Varying degrees of automation are also available. For full automation, all that is required is to load a sample tray with template DNA; the equipment performs the labeling and analysis. The other option is to perform the labeling reactions with fluorescent dyes, load the samples onto a gel, and place the gel into the DNA sequencer. The equipment performs the separation and analysis. The system automatically identifies the nucleotide sequence and saves the information on the computer. Thus, only a review of the data is necessary to ensure no anomalies were misidentified by the computer. The greatest obstacle to researchers when converting from manual to automatic DNA sequencing is being required to learn the use of computer software necessary to interpret the results. 9. steps to sequencing a bacterial genome: whole genome “shot-gun cloning” method 1. DNA is randomly sheared into small fragments that are then ligated into plasmids 1. thousands of plasmids are generated, each of which has some fragment of the genome you want to sequence 2. this “library” of plasmids are put into E. coli so that each may be purified in amounts sufficient for sequencing (each colony of E. coli has a diff plasmid) 3. the plasmids are isolated from the thousands of E. coli clones each harboring a plasmid w a different random segment of the genome 4. determine 500-1000 bp of sequence from each end of each cloned piece of DNA 2. this is possible by using primers complementary to the plasmid area flanking either end of the insert 5. after sequencing thousands of plasmids this way, the sequences can be stitched together by a computer to assemble the full genome 2. limitations: 1. cloning bias – some segments will be toxic to E. coli & cannot be cloned 1. this will result in “gaps” in the assembled sequence that must be filled in w more laborious sequencing methods 2. project maintenance issues – must maintain a lot (several thousands) of E. coli isolates, each of which must be grown up, frozen down for storage, re- grown when the plasmid is to be purified, maintained in a database, etc. 3. slow & expensive – even the most advanced & high-throughput Sanger sequencing platforms yield <100 kilobases per fun 2. a typical genome is 5 million bps 3. if you want good coverage of a genome using this random method, you need a lot of runs PYROSEQUENCING 10. real-time sequencing: you get the sequence as the rxn progresses 11. the rxn can be performed in liquid or w the DNA substrate attached to a slide/bead 12. does not involve electrophoresis 1. instead, the readout is light production that is measured by a camera/detector 2. the light is generated by a combination of enzymes when a nucleotide is incorporated during chain extension 13. start: primer is annealed to template (similar to Sanger method) 14.
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