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Genomics seminar
- 1. Genomics Presented by: Samaneh.Rasoulinejad Fall 2013 - 2014
- 2. What is Genomics? • • Genomcis is the study of all genes present in an organism In 1986 mouse geneticist Thomas Roderick used Genomics for “mapping, sequencing and characterizing genomes”
- 3. Introduction • Genomics built on recombinant-DNA technology (developed since early 1970s) • Thorough understanding of recombinant-DNA techniques • Prerequisite for understanding genomics technologies • Differences between genomics and recombinant-DNA technology • Genomics is high throughput approaches to allow more analyses in parallel • Genomics is dependent on computational analysis due to larger data sets
- 4. Sequence the entire genome by cutting it into small, manageable pieces (fragments) Assemble the entire genome from the pieces (fragments) Make sense of the genome Understand how gene expression takes place? How life processes are networked? Understand life??
- 5. Technical Foundations of Genomic and cDNA libraries Genomics DNA Hybridization and Northern blots Subcloning in vectors Restriction-enzyme mapping DNA sequencing PCR amplification Genomics and Medicine
- 6. What we hope to gain from genomics -Drug, diagnostics, and prognostics development - Genotyping to predict patient susceptibility to disease - Personalized healthcare based on an individual’s genomic features genome decision support systems genotype molecular profile patient history knowledge base drugs diagnostics prognostics health
- 7. Over 1,000 disease genes were characterized by 2000
- 8. How to make a genomic library ori total genomic DNA ampR genomic DNA restriction enzyme anneal and ligate ori ampR ori plasmid (black) ampR ori ampR ori ampR same restriction enzyme transform E. coli; select for Amp resistance
- 9. selected colonies tissue or cell membrane mRNA polyA stationary support polyT Radioactive probe plasmid E. Coli bacteria hybridization X-ray film cDNA library Clone 1 2 3 4 5
- 11. Microarrays • Basis of microarrays for determining gene expression • Process by which complementary strands find each other • A–T and C–G base pairing • speed and fidelity: dependent on temperature, salt, sequence, and concentration (High temp and low salt)
- 12. • • • • Microarrays permit the simultaneous analysis of the RNA expression of thousands of genes. For fully sequenced genomes, microarrays can be used to analyze the expression of every gene. Prior to the introduction of microarrays, RNA abundance was usually analyzed through hybridization to RNA bound to filters. These Northern blots normally had no more than 20–40 lanes, and no more than three probes could be used simultaneously. In contrast, microarrays can interrogate 30,000 genes at the same time, vastly increasing our ability to analyze RNA expression.
- 13. Northern blot and microarray 0 2 5 6 7 hrs 0 2 5 6 7 9 11 hrs DMC1 – DMC1 – SPS1 – DIT1 – SPS1 – SPS100 – 0 2 5 6 7 9 11 hrs DIT1 – SPS100 – fold repressed fold induced >20 10x 3x | 3x 10x >20 1:1 Identify genes whose expression was induced during sporulation in yeast
- 14. Cross-hybridization • • • • Hybridization to a related, but not identical, sequence = cross-hybridization Example: A probe from one member of a gene family is likely to hybridize to all other members Problem in microarrays, particularly cDNA arrays Oligonucleotide arrays prescreened to eliminate sequences likely to cross-hybridize
- 15. Improved disease diagnostics from genomics • Microarray analysis of gene expression from four different types of tumors
- 16. Microarrays and cancer • • Histology not always effective tool for prognosis and diagnosis Microarrays distinguish cancerous tissues on the basis of a gene expression profile • Use in diagnosis (presence) • • Example: characterizing acute lymphoblastic leukemia. Also breast cancer. Use in prognosis • Example: assessing the likelihood of metastasis in medulloblastoma (brain tumor in children)
- 17. Microarrays in the prognosis of metastasis (childhood cancer: medulloblastoma) • • • • Identified 85 genes with different levels of expression in metastatic (M+) and nonmetastatic tumors (M_) 59 up and 26 down 72% accuracy in predicting metastasis Identified genes induced in metastasis • Could serve as potential drug targets for in vitro experiments • platelet derived growth factor receptor alpha (PDGFRα). Antibodies prevent migration. M– M+ green = down regulated red = up regulated
- 18. Cancer genome projects One in three people will suffer from cancer in his or her lifetime. Cancer Genome Anatomy Project (CGAP) Established 1997 by National Cancer Institute (USA) Specializes in EST sequencing Human Cancer Genome Project (HCGP) Established 1999 by Brazilian research groups Cancer Genome Project (CGP) Established 2000 by Wellcome Trust and Sanger Institute (United Kingdom) Specializes in genomic mutations leading to cancer Funding: $15 million to $60 million
- 19. Classes of microarrays • Custom/spotted/two-color microarrays (cDNAs, BACs) • High-density oligonucleotide arrays (GeneChip, Affymetrix) • Long oligonucleotide microarrays - Agilent (25-60 bases) - Illumina (50 bases) - Nimblegen (50-75 bases)
- 20. Affymetrix oligonucleotide arrays The array elements are a series of 25mer oligos designed from known sequence and synthesized Directly on the surface The entire array is formed by >500,000 cells, each containing a different oligo
- 21. subcloning • Propagating fragments of cloned DNA • Used for sequencing and protein production • Plasmid vectors • Replicate in bacteria • Resistant to antibiotics • Cloning sites ORI Region into which DNA can be inserted Plasmid cloning vector ampr
- 22. Subcloning: vector and fragment • Vector and fragment to be DNA fragment inserted must have compatible ends • Sticky ends anneal • Enzyme ligase makes covalent cloning vector bond between vector and fragment • Use of recombination instead of restriction sites recombinant plasmid restriction enzymes
- 23. Recombination cloning • • • • Uses site-specific recombination for subcloning DNA fragment flanked by recombination sites Add recombinase “Clonase®” Moves fragment from one vector to another
- 24. DNA sequencing • Most current sequencing projects use the chain termination method • • Based on action of DNA polymerase • • Also known as Sanger sequencing, after its inventor, Fredrick Sanger Adds nucleotides to complementary strand Requires template DNA and primer
- 26. Chain-termination sequencing • Dideoxynucleotides stop synthesis • Chain terminators Included in amounts so as to terminate every time the base appears in the template • Use four reactions • • One for each base: A,C,G, and T
- 28. Sequence detection To detect products of sequencing reaction Include labeled nucleotides Formerly, radioactive labels were used Now fluorescent labels Use different fluorescent tag for each nucleotide Can run all four reactions in same lane
- 29. Pyrosequencing • • • based on the sequencing by synthesis principle. it relies on the detection of pyrophosphate release on nucleotide incorporation The technique was developed by Mostafa Ronaghi and Pål Nyrén at the Royal Institute of Technology in Stockholm in 1996
- 32. PCRs • • • • • • • • • • Colony PCR Helicase PCR Hot- start In situ Intersequence specific Inverse Multiplex Quantitative Touch down ......
- 33. Resources • Bioinformatics, Genomics, and Proteomics (Ann Finney Batiza, Ph.D.) • SciencePages • Functional Genomics (Michael Kaufmann and Claudia Klinger Private Universitt, Witten/Herdecke gGmbH, Witten, Germany) Introduction to Genomics by Arthur M. Lesk, 2007, Oxford University Press
- 34. Thank you