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MSU MMG 301 - Lecture 18

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Lecture 181. Explain how the Sanger dideoxy sequencing method works a. 1st Generation:i. DNA polymerase reactions on cloned DNA fragments using chain-terminating basesii. Resulting fragments separated by capillary electrophoresis iii. Each dideoxynucleotide is labeled with its own fluorescent dye2. Explain the shotgun sequencing approach for genome sequencing a. Shotgun- Genomic DNA is fragmented into pieces and fragments are cloned into plasmids before sequencing. b. Steps:i. Genomic DNA purificationii. Genomic DNA fragmented into small piecesiii. Fragments cloned into plasmids and randomly sequenced iv. Overlapping sequences are aligned using computersv. Assembly of consensus sequence3. Compare and contrast the two DNA sequencing methods a. 1st Generation:i. DNA polymerase reactions on cloned DNA fragments using chain-terminating basesii. Resulting fragments separated by capillary electrophoresis b. 2nd Generation (next gen):i. Massively parallel methods: thousands of simultaneous sequencingreactionsii. Uses light emission to detect which nucleotide is incorporated during sequencingiii. Method development aided by miniaturization of reactions, robotics,and improvements in computing poweriv. Sequence is read at the same time as DNA is synthesized v. NO DDNTP’s4. Explain how genome assembly works a. Organizing the sequences in the correct order, eliminating overlaps and gapsb. Sequence alignments and formation of contigsc. Computer software aligns all the sequencesd. Sequence overlaps indicate order of fragmantse. Software eliminates overlaps and creates final sequence = contig5. Compare and contrast contigs, scaffolds, and closed genomes a. Contigi. Not connected there are gaps betweenb. Scaffoldi. Gap of unknown sequences between to connectc. Closed genomesi. All connected and no unknown sequence gapsii. Takes much longeriii. Know entire sequences6. Explain why many genome sequencing projects stop at the draft genome stage a. Because shotgun sequencing and assembly are heavily automated, but gap closure is not. A closed genome is much more expensive and time consuming to generate than a draft genome seqeunce7. Explain what defines an open reading frame (ORF) a. A start codonb. A stop codonc. A ribosome binding site (RBS) in front of the start codond. Correct codon usage statistics 8. Explain the concept of codon bias in a given organism and how it is used in gene prediction a. Some codons are greatly preferred over others even though they encode the same amino acidb. Gene predictioni. Computer finds possible start codonsii. Computer finds possible stop codonsiii. Computer counts codons in between start and stopiv. Computer finds possible RBSv. Computer calculates codon bias in ORFvi. Computer decides if ORF is likely to be genuinevii. List of possible ORFs9. Explain why an ORF encodes a protein, and not an RNA molecule a. rRNA is in a ORF and RNA codes for proteins not another RNA molecule and DNA is not present10. Explain how the metabolism of uncultured bacteria can be predicted from their genome sequence a. You can predict metabolic pathwaysb. Cant grow in the lab, need nutrients from another organisms, so they took a single bacterial symbiont from the eukaryotes and sequenced them. That is how they got what the genes did by sequencing to predict the pathways.11. Compare and contrast microbial genomics and metagenomics a. Microbial genomicsb. Metagenomicsi. Communities containing uncultured organisms that are analyzed bygenome and/or RNA sequencing.ii. Human genome 22,000 genesiii. Human microbiome ~ 8 million genesiv. Can analyze microbial communities found in interesting environments, such as:1. Sargasso sea2. Human gut3. Clouds4. Dirty shower curtains12. Give examples of applications of metagenomics a. Analyze microbial communities in interesting situations (dirty shower curtain), compare community composition effect of disease/ diet on humanmicro biome effect of climate change/ pollution on soil microbial community13. Explain what the transcriptome is a. Definition: Quantifies gene expression based on the abundance of each RNA in a cell.b. RNA sequencing = whole transcriptome14. Explain how RNA sequencing is used to identify which genes are expressed in a bacterial culture a. Sequence of total RNA aligned with genome sequence b. 1. Quantifies transcription level of every genec. 2. Discovery of new genes15. Give examples of applications of transcriptomics a. Microbiologyi. To identify metabolic pathways needed to grow in certain environmentsii. To determine the function of individual bacteria in a mixed microbialpopulationiii. To identify genes involved in bacterial pathogenicity/virulence b. Health/Medicinei. Comparison of gene expression in cancer cells vs healthy cellsii. Comparison of gene expression in different human


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MSU MMG 301 - Lecture 18

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