Lecture Slides on Assembly Software, 2D-Gels | CAP 5510, Study Guides, Projects, Research of Computer Science

Download Lecture Slides on Assembly Software, 2D-Gels | CAP 5510 and more Study Guides, Projects, Research Computer Science in PDF only on Docsity! 3/27/08 CAP5510 1 CAP 5510: Introduction to Bioinformatics Giri Narasimhan ECS 254; Phone: x3748 giri@cis.fiu.edu www.cis.fiu.edu/~giri/teach/BioinfS08.html 3/27/08 CAP5510 2 Sequencing (Review by Shendure et al., Nat. Rev. Gen. 2004) Goal: ULCS (Ultra low cost sequencing) Personal genome project Microelectrophoretic sequencing Electrophoretic separation with single-base resolution Microfabrication, multiplexing, miniaturization technology Hybridization sequencing Differential hybridization with short probes Cyclic-array sequencing of amplified molecules Multiple cycles of manipulation of spatially separated fragments 2 Types: Pyrosequencing (454 Sequencing) fluorescent in situ sequencing (Solexa and Polony sequencing) Non-cyclical, single molecule, real-time methods DNA sent through nanopore & differences in pore conductance measured 3/27/08 CAP5510 5 2D Gel Electrophoresis 3/27/08 CAP5510 6 2D-Gels First Dimension Methodology of a 2D Gel: Denatured cell extract layered on a glass tube filled with polyacrylamide saturated with solution of ampholytes, a mixture of polyanionic[(-) charged] and polycationic [(+) charged] molecules. When placed in an electric field, the ampholytes separate and form continuous gradient based on net charge. Highly polyanionic ampholytes will collect at one end of tube, highly polycationic ampholytes will collect at other end. Gradient of ampholytes establishes pH gradient. Charged proteins migrate through gradient until they reach their pI, or isoelectric point, the pH at which the net charge of the protein is zero. This resolves proteins that differ by only one charge. Entering the Second Dimension: Proteins that were separated on IEF gel are next separated in the second dimension based on their molecular weights. The IEF gel is extruded from tube and placed lengthwise in alignment with second polyacrylamide gel slab saturated with SDS. When an electric field is imposed, the proteins migrate from IEF gel into SDS slab gel and then separate according to mass. Sequential resolution of proteins by their charge and mass can give excellent separation of cellular proteins. As many as 1000 proteins can be resolved simultaneously. *Some information was taken from Lodish et al. Molecular Cell Biology. 3/27/08 CAP5510 7 2D-gels Comparing Proteomes For Differences in Protein Expression Comparing Different Sample Types For Changes in Protein Levels 3/27/08 CAP5510 10 Genomics Study of all genes in a genome, or comparison of whole genomes. Whole genome sequencing Whole genome annotation & Functional genomics Whole genome comparison PipMaker: uses BLASTZ to compare very long sequences (> 2Mb); http://www.cse.psu.edu/pipmaker/ Mummer: used for comparing long microbial sequences (uses Suffix trees!) 3/27/08 CAP5510 11 Genomics Study of all genes in a genome Gene Expression Microarray experiments & analysis Probe design (CODEHOP) Array image analysis (CrazyQuant) Identifying genes with significant changes (SAM) Clustering 3/27/08 CAP5510 12 Comparative Genomics Comparison of whole genomes. Whole genome sequencing Whole genome annotation & Functional genomics Whole genome comparison PipMaker, MultiPipMaker, EnteriX: PipMaker uses BLASTZ to compare very long sequences (> 2Mb); http://www.cse.psu.edu/pipmaker/ Mummer: used for comparing long microbial sequences (uses Suffix trees!) Many more! = ¥: “n ‘= a. ~ * * a: ‘ -“ Toate ; 7 Te ae 10-| * ele a ne T 1 T T T —— sal pl 45 5.0 5.5 6.0 6.5 6.07.5 TRENDS in Biotechnology 3/27/08 CAP5510 15 3/27/08 CAP5510 16 Other Proteomics Tools From ExPASy/SWISS-PROT: AACompIdent identify proteins from aa composition [Input: aa composition, isoelectric point, mol wt., etc. Output: proteins from DB] AACompSim compares proteins aa composition with other proteins MultIdent uses mol wt., mass fingerprints, etc. to identify proteins PeptIdent compares experimentally determined mass fingerprints with theoretically determined ones for all proteins FindMod predicts post-translational modifications based on mass difference between experimental and theoretical mass fingerprints. PeptideMass theoretical mass fingerprint for a given protein. GlycoMod predicts oligosaccharide modifications from mass difference TGREASE calculates hydrophobicity of protein along its length 3/27/08 CAP5510 17 Gene Networks & Pathways Genes & Proteins act in concert and therefore form a complex network of dependencies. 3/27/08 CAP5510 20 STSs and ESTs Sequence-Tagged Site: short, unique sequence Expressed Sequence Tag: short, unique sequence from a coding region 1991: 609 ESTs [Adams et al.] June 2000: 4.6 million in dbEST Genome sequencing center at St. Louis produce 20,000 ESTs per week. 3/27/08 CAP5510 21 What Are ESTs and How Are They Made? Small pieces of DNA sequence (usually 200 - 500 nucleotides) of low quality. Extract mRNA from cells, tissues, or organs and sequence either end. Reverse transcribe to get cDNA (5’ EST and 3’EST) and deposit in EST library. Used as "tags" or markers for that gene. Can be used to identify similar genes from other organisms (Complications: variations among organisms, variations in genome size, presence or absence of introns). 5’ ESTs tend to be more useful (cross-species conservation), 3’ EST often in UTR. 3/27/08 CAP5510 22 DNA Markers Uniquely identifiable DNA segments. Short, <500 nucleotides. Layout of these markers give a map of genome. Markers may be polymorphic (variations among individuals). Polymorphism gives rise to alleles. Found by PCR assays.