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Topic 10: Microbial Genomics - Fall 2013

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BIOL 240
Josh Neufeld

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BIOL 240 Fall 2013 Topic 10: Microbial Genomics Genomics: discipline involving mapping, sequencing, analyzing and comparing genomes Functional genomics: assigning function to unknown genes by analyzing biochemical and physiological ▯ ▯ effects of mutants Proteomics: analysis of the structure, function and regulation of proteins Transcriptomics: analysis of the expression of all of the transcripts in the genome at once Metagenomics: extraction and analysis of DNA directly from an environmental sample Sanger/Dideoxy DNA sequencing • takes advantage of primer specific DNA synthesis activity of DNA polymerase • steps: • cloning the gene fragment of interest • DNA synthesis • isolate the plasmid containing the cloned DNA and denature to provide a single-stranded template • add radiolabeled short oligonucleotide primer, DNA polymerase, and the four deoxyribonucleotide triphosphates (dNTPs - N refers to the bases G, C, T and A) • primer is complementary to the vector sequence - it anneals to the single-stranded template, providing a base-paired free 3’-OH to which DNA polymerase can add additional nucleotides • as DNA polymerase adds nucleotides, it makes a piece of DNA complementary to the single-stranded template • split the mixture into four different aliquots • add different ddNTP (dideooxynucleotides) into the mixtures (ddGTP, ddATP, ddTTP, and ddCTP) to terminate the elongation reaction once a dideoxynucleotide becomes incorporated no 3’-OH is available for the addition • of the next dNTP • DNA products of different lengths are formed in the aliquot each defined by the site of insertion of the ddNTP • the four reaction sets are denatured to release the newly radiolabeled strands from the unlabeled template strands • electrophoresis • the mixture is separated by electrophoresis by a polyacrylamide gel - can discern fragments differing in length by a single nucleotide • gel is exposed to X-ray - radiolabeled fragments produce a ladder-like pattern - read to give DNA sequence • initially this technique resolved 100 nucleotides in a single run • the use of fluorescent dyes instead of radioactive labels eliminates the X-ray step - products can be detected as they migrate past a laser on near the end of the gel - eliminates the need to carry out four reactions and run four samples on the gel fluorescent = safer, cheaper and easier • • reading sequences no longer done manually - “base-calling” computer programs used Automated/cycle-sequencing methods: larger DNA fragments undergo larger electrophoretic ▯ ▯ ▯ separation - obtain sequences of 700-1000 nucleotides in hours Primer walking: longer sequences obtained using repeated rounds of sequencing with primers ▯ ▯ complementary to the end of the last segment sequenced • sequence obtained from first run can be used to design a second primer that when subjected to a second run will anneal farther along the fragment • consecutive runs can allow sequencing of the entire fragment most cloning plasmids can hold around 10 kb of cloned DNA, cosmids can hold around 45 kb of DNA • i Pyrosequencing • like Sanger, relies on DNA polymerase synthesis of the complementary strand of a single-stranded template • differs from Sanger in the fact that instead of using ddNTPs, the four dNTPs are added one at a time • if the dNTP is complementary to the template base, it is incorporated into the growing piece of DNA which releases pyrophosphate which can converted by ATP-sulfurylase to ATP • ATP drives a luciferase reaction, which produces light - detected with a charge-coupled device (CCD) camera • faster and cheaper than Sanger - no DNA cloning • 454 DNA sequencing - DNA does not have to be cloned into vectors • DNA fragmented into short pieces and ligated with adapters that facilitate the trapping of the fragments on separate beads • each bead contains a single, short DNA fragment which can be separated into its own droplet of PCR reaction within an oil emulsion - PCR is carried out - results in millions of DNA molecules in each bead • these beads are distributed into the wells of a flow cell picotiter plate (a slide that almost has 2 million 75-picotiter-volume wells) • each well contains a single bead and its bound DNA • DNA sequencing reagents added - reactions carried out by addition of each of the four dNTPs by horizontal flow over the wells • CCD camera captures the light • over 500 million bases of sequencing data can be generated from a single machine in a 10-hour run • Illumina sequencing - DNA polymerase adds fluorescently labeled nucleotides to amplified, immobilized fragments of DNA after imaging, the fluorescent label is cleaved from the terminal nucleotide • • another fluorescent nucleotide is added • Ion torrent sequencing • sensor detects hydrogen ions that are released during the synthesis of
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