First Prelim Thursday February 18 in class Material included - lecture of 2.11.10 A - Mariano go to PLS 233 McConnell - Z go to RRB125 Reviews on Monday 4:30 - 6:00 pm Warren 245 Wednesday 5:00 - 6:30 pm Warren 131 Bases, nucleosides and nucleotides Chemical and physical properties of nucleic acids - acid and base hydrolysis, UV absorption, Tm, denaturation and renaturation Structure of DNA - ABZ, palindrome, triplex and G4 DNA Genomes - complexities and comparative genomics DNA based technologies Cot curve Recombinant DNA Cloning methods and vectors Enzymes - nucleases, restriction endonucleases, ligases First Prelim - Please bring a simple, non-graphing calculator The most challenging task in the genome project was not in finding all the pieces of Humpty The challenge was how to put Humpty together again Ordering of the clones in a DNA library using STS (sequence tagged sites) or EST (expression sequence tag) STS sequence-tagged sites Problem - Ch 9: 9 } contigs 100-300kb Proteomics - the study of the function of proteins at the scale of a whole cell. Determining function based on interacting partners or ?guilt by association?. Two-hybrid analysis Protein chips Purification of protein complexes Mass spectrometry (MALDI - matrix-assisted laser desorption/ionization mass spectrometry) p. 100 Box 3-2 Functional genomics - study of the function of genes in a whole genome scale DNA microarrays (examine the changes in gene expression across the entire genome in different mutant alleles, under different developmental conditions, of disease states, or upon drug treatment.) Whole genome deletion analysis - identify function of every gene in the genome by systematic deletion. (18.2% of genes in the haploid yeast genome are essential.) DNA Polymerase-based Technologies for studying functional genomics DNA sequencing Frederick Sanger won two nobel prizes in chemistry 1958 amino acid sequencing of insulin 1980 DNA sequencing (shared with Walter Gilbert) Problem: Ch 8:12 Study guide: p.108 #6 DNA sequencing - Sanger?s (dideoxy) method terminator DNA polymerase GATTCGAGCTGA large small Radioactively labeled at 5?-end 4 RXs each containing a different ddNTP High resolution gel system Automated sequencing reactions - each reaction can resolve 600 to 750 bp (labeled with fluorescent dyes) Microarray analysis of whole genome expression: Sequence of entire genome is known -PCR amplify each open reading frame (ORF) Renaturation kinetics Competitive hybridization -making cDNA from mRNA (unstable) Polymerase chain reaction or PCR Kary Mullin, Nobel prize in chemistry 1993 amplifies a specific sequence from a minute amount of DNA template in a short time 1. DNA template - small amount 2. Oligonucleotide primers - in vast molar excess 3. A thermostable DNA polymerase Problems: Ch.9: 5, 6 Polymerase Chain Reaction - PCR 225 5? 3? AAAAA TTTTT 3? 5? 5? 3? AAAAA mRNA cDNA Reverse transcriptase Preparation of cDNA for microarray analysis of whole genome expression TTTTT 3? 5? alkali mRNA Genome-wide expression studies - microarray analysis A survey of the expression pattern of all genes within the genome under a particular condition All ORFs are PCR amplified and gridded on a slide (6000 ORFs on one slide for the S. cerevisiae genome The level of expression of each gene is measured by hybridizing cDNAs to the ORFs of the entire genome vegetative sporulating 6200 genes deposited in 6200 spots on a glass slide. Each spot represents a different gene. Competitive hybridization Relative expression sporulation/vegetative: = higher than in veg cells = same level of expression = lower than in veg. cells Fluorescently labeled Genome chips Viewed under a Laser scanner See fig 9-22 for additional example. Photolithography This technique for preparing DNA microarray makes use of nucleotide precursors that are activated by light, joining one nucleotide to the next in a photoreaction. Synthesize array on slide. 3?-OH is blocked and can be activated by light Polynucleotides of 25 long are sufficient to specify unique sequence information
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