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  • This technique makes it possible to amplify a specific sequence of DNA from a complex mixture (for example, one gene from within an entire genome or one cDNA from within the entire repertoire of cellular mRNA). The investigator can begin with a single copy of a gene or mRNA and, in a few hours, have billions of copies. Today PCR is used in almost every aspect of molecular biology and molecular medicine that involves DNA including rapid identification of pathogens, diagnosis of inherited and microbial diseases, and monitoring disease progression and response to drug treatment. <br /> The essential ingredients are the DNA molecule to be copied (the template); two oligonucleotide primers, each with a sequence complimentary to a region of about 20 bases on one side of the region to be amplified; Taq polymerase; and all four dNTPs. <br /> CLICKs <br />
  • One recent application that is gaining popularity is using PCR to quantitate the number of copies of a gene in cells. In the clinic this method is used when assessing amplification of genes such as HER2/Neu in breast cancer cells and multidrug resistance genes in other types of cancer cells, and for diagnosing and monitoring viral infections such as CMV and EBV. <br />

Brian_Strahl 2013_class_on_genomics_and_proteomics Brian_Strahl 2013_class_on_genomics_and_proteomics Presentation Transcript

  • Genomics and Proteomics Brian Strahl 2013 1
  • Outline I. Background and history of Genomics • How did the genomics field come about? • Key molecular biology advances • Brief review of Epigenetics I. Genomic methods and their applications in Medicine • Basic techniques used in genomics methods • Microarray technologies • Genomic tests currently used in the clinical setting • High-throughput genomic sequencing I. Proteomics and and their applications in Medicine • Proteomic platforms and clinical uses • Mass spectrometry and biomarker discovery in human diseases 2
  • What is Genomics? “The study of DNA sequences, genes, and genome organization and function” The word “Genomics” is most commonly used to describe any form of high-throughput and/or largescale approach towards understanding gene and genome function 3 View slide
  • Why should I care to learn about genomics and proteomics? How will this knowledge help me in the clinic someday? Biomedical researchers use genomics to: •Determine the extent of genetic variation between individuals •Understand disease etiology (causes) •Distinguish disease/cancer subtypes and categories •Identify new diagnostic biomarkers •Facilitate faster drug development (pharmacogenomics)*discussed by Dr. Mcleod •Reveal new therapeutic targets The outcome of this biomedical research will allow the average physician to: •Determine an individual’s predispositions to certain diseases or illnesses •Diagnose disease and cancer subtypes •Predict patient outcome, disease recurrence and treatment strategies •Individualize (or tailor) patient treatments 4 View slide
  • The Genomics Revolution How did it begin? • DNA structure and the genetic code 1950’s • Recombinant DNA technology • Polymerase Chain Reaction 1970’s/1980’s • Automated DNA sequencing •The Human Genome Project 1990’s 5
  • Epigenetics However, genetic diversity is also regulated by events outside of the DNA! 1. DNA information • The genetic code - i.e. triplicate base pairs in genes that codes for a particular amino acid. 2. Epigenetic information • Effects on gene or genome function that are not specified in the DNA sequence itself (e.g. histone post-translational modifications). 6
  • Organization of Eukaryotic Chromatin DNA double helix Histone H3 H2B H2A H4 Nucleosomes Solenoid Chromatin loop: Chromatin ~100,000 bp DNA 7
  • Epigenetic regulators 1. Chromatin remodeling complexes (e.g. Swi/Snf) 2. Histone modifications Acetylation Phosphorylation Methylation Ubiquitylation Sumoylation 3. Histone variants (e.g. H2A.Z, CENP-A, etc.) 4. DNA methylation 8
  • Outline I. Background and history of Genomics • How did the genomics field come about? • Key molecular biology advances • Brief review of Epigenetics I. Genomic methods and their applications in Medicine • Basic techniques used in genomics methods • Microarray technologies • Genomic tests currently used in the clinical setting • High-throughput genomic sequencing I. Proteomics and and their applications in Medicine • Proteomic platforms and clinical uses • Mass spectrometry and biomarker discovery in human diseases 9
  • Basic techniques used in genomics methods 10
  • Key methods in Genomics: I. Blotting and Hybridization Southern blots Northern blots western blots DNA RNA Protein 1. Separate molecules by electrophoresis according to size 2. Transfer to solid support (membrane) 3. Probe for specific sequence (hybridization for nucleic acid; antibodies for proteins) 11
  • Key methods in Genomics: I. Blotting and Hybridization Southern blots Target: Genomic DNA Goal: to examine gene structure, organization, size and copy number Northern blots Target: RNA Goal: to examine gene expression western blots Target: Protein Goal: to examine protein levels and their post-translational modifications (e.g. phosphorylation) 12
  • Southern blot procedure: Probing the membrane Blot after transfer, UV light, 80oC X-ray film after exposure to blot Blot washed to remove excess, probe Blot with radioactive probe Hot probe! Complementary base pairing 13
  • Southern blot Application Example showing increased numbers (amplification) of the Her2/neu gene in breast cancers 2-5 Single copies copy 5-20 copies >20 copies 14
  • Western blots: General procedure Electroblotting tank Power supply _ + Proteins transferred to membrane Gel after electrophoresis Visualize using X-ray film Incubate membrane with antibody against protein of interest Detect bound antibody 15
  • Western blots: Clinical applications Clinical Example of an HIV test using a Western blot Band pattern Interpretation Lane 1, HIV+ serum (positive control) Lane 2, HIV- serum (negative control) Lane A, Patient A Lane B, Patient B Lane C, Patient C 16
  • Northern blots: Applications o Transcript size o Gene regulation o Alternate transcripts o Transcript (expression) levels o Related transcripts Ex: induction of Fos mRNA after growth stimulation RNA from unstimulated cells RNA from cells stimulated for: RNA from tumor cells 15” 30” 60” Fos mRNA control mRNA X-ray film 17
  • The Polymerase Chain Reaction 1 copy Double-stranded DNA 3’ 5’ 5’ 3’ Primers complimentary to ends of target region 35 cycles yields 235 = 34.4 billion copies Repeat denature, anneal, & extend + 5’ 3’ Heat denature strands, cool to anneal primers 5’ 3’ 3’ 4 copies 5’ Thermo-stable DNA polymerase 3’ 5’ Extend extend primers 5’ 5’ 3’ 2 copies 3’ 5’ 3’ 3’ 5’ Denature, anneal 18
  • Reverse Transcriptase (RT)-PCR: Detecting Specific mRNAs PCR can also be used to detect specific mRNAs via amplification of Reverse Transcriptase-derived cDNA (RT-PCR) RT-PCR mRNA: TTTTTTTTTT 5’ dT primer AAAAAAAAAA HO + 1. Reverse Transcriptase (RT) cDNA: PCR DNA: etc 19
  • Real-time PCR: Towards High-throughput Quantitation of DNA & RNA oligonucleotide complimentary to target Quencher Fluorescent Reporter Dye + target DNA + primers heat and anneal 3’ 5’ 3’ 5’ 3’ 5’ Taq polymerase 3’ 5’ Fig from: www.hgbiochip.com/images/QuantitativePCR.gif Repeat, measure fluorescence at each cycle 3’ 5’ 3’ 5’ Fluorescence no longer quenched! 20
  • Some Clinical Applications of Real-time PCR: Genomic PCR o Determining the number of gene copies (e.g. HER2) RT-PCR o Quantitating the level of RNA viral infection (e.g. HIV) o Comparing expression of suspect genes in normal versus diseased tissues or tumors (e.g. p53, RB, ER) 21
  • High-through put genomic technologies 22
  • Microarrays: gene expression profiling Genome wide expression (“Transcriptome”) analysis o Emphasis on global changes instead of single genes o Towards compiling atlases of genome expression Important clinical impact, including: o Understanding disease etiology o Distinguishing disease subtypes (molecular signatures) o Identifying new diagnostic markers o Revealing new therapeutic targets 23
  • Two-color cDNA Microarrays: A Comparative Analysis Approach: compare levels of expression of individual genes in a large population between test and reference samples . . . Cancer cell Make cDNA pools using nucleotides tagged with red (Cy5) or green (Cy3) fluorescent dye Normal cell Here: Test = cancer cell Ref = normal cell mRNAs cDNAs 24
  • Outline of the Two Color – cDNA Microarray Approach Cancer cell Normal cell Combine cDNAs Hybridize to array Wash, excite with lasers Cy5/Cy3 0.03 50 Record emissions w/ detectors 1 Gene exp higher in cancer cell Gene exp lower in cancer cell Gene exp similar in cancer cell 25
  • Microarray Technology 26
  • Data Selection & Clustering: Making Sense Out of Arrays Original data Clustered data Genes inhibited quickly Inhibited after lag, then induced Induced after lag 123456 Time 123456 Induced after longer lag Inhibited Induced Time 27
  • Gene Arrays: Distinguishing Different Cancers Mesenchymal Leukemia Epithelial Distinct gene expression profiles mark different cancers, help distinguish primary tumor Melanoma 28
  • Gene Arrays: Distinguishing Different Disease Subtypes Tumor groupings Probability The 5 tumor types: HER2+ Basal tumors Survival months 29
  • Gene Arrays: Distinguishing Different Disease Subtypes Example 2: Distinguishing acute myeloid leukemia (AML) from acute lymphoblastoid leukemia (ALL) These two cancer types are difficult to distinguish using traditional cytological, cytogenetic and biochemical assays. This is relevant as while daunorubicin and cytarabine work best for AML, vincristine and methotrexate works better for ALL 30
  • Gene Arrays: use in the clinic Several FDA approved genetic tests that are available to you now: 1) AmpliChip Cytochrome P450 Genotyping test • Looks for common genetic mutations and/or variations that might occur in the cytochrome P450 enzymes CYP2D6 and CYP2C19. These genes regulate drug metabolism in the liver. Test indicates whether there is a likelihood of certain drugs to be metabolized faster or slower. 1) MammaPrint® test • Examines the gene signature of 70 cancer-related genes commonly activated in breast tumors. The test determines the likelihood that a breast cancer will return within 5 to 10 years. A score is given that determines whether the patient is at “low risk” or “high risk” for tumor recurrence. Such information will help guide doctors in the appropriate treatment and follow-up care for each 31 individual patient.
  • MamaPrint study 295 patients 32 N Engl J Med, Vol. 347, 2002 p1999
  • Next Generation DNA Sequencing Technologies Why bother? Provides rapid and extensive sequence information at a very low price What can you do with this technology? 1) Sequence a human genome in two weeks Obtain SNPs, insertions and deletions (“Indels”) 1) Rapidly obtain the “transcriptome” (RNA-Seq) RNA  cDNA  High-throughput sequencing 1) Obtain transcription and chromatin factor binding sites (ChIP-Seq) Antibody immunoprecipitation, isolate DNA, sequence 33
  • Comparing Sequencers Roche (454) Illumina GAIIx SOLiD 4 Chemistry Pyrosequencing Polymerase-based Ligation-based Amplification Emulsion PCR Bridge Amp Emulsion PCR Paired ends/sep Yes/3kb Yes/400 bp or 5000kb Yes/200bp or 3-10 kb Mb/run 100 Mb 48 Gb 100 Gb Time/run 7h 8 days 12 days Read length 450 bp 76bp 50 bp Cost per run (total) 1/16 plate $562 (36bp) $700 N/A The human genome contains about 3,400 megabases (3.4 GB) Illumina has now lowered the cost of its individual sequencing service to ~$5000/sample. They are offering a discounted price for people with serious medical conditions who could potentially benefit from having their genomes decoded. 34
  • Reconstructing Genome Sequence reads Genomic DNA 35
  • Challenges with Fragment Assembly • Sequencing errors can happen! ~1-2% of bases are wrong • Repeat regions are difficult False overlap due to repeat 36
  • Outline I. Background and history of Genomics • How did the genomics field come about? • Key molecular biology advances • The Human Genome Project and its discoveries/impact • Brief review of Epigenetics I. Genomic methods and their applications in Medicine • Basic techniques used in genomics methods • Microarray technologies • Genomic tests currently used in the clinical setting • High-throughput genomic sequencing I. Proteomics and and their applications in Medicine • Proteomic platforms and clinical uses • Mass spectrometry and biomarker discovery in human diseases 37
  • Proteomics: A More Challenging Undertaking Very challenging: o Proteins are inherently more diverse than DNA/RNA o Tip of the iceberg: numerous covalent modifications o Difficult to cleanly separate different cell compartments and cell types Ca nc B C Phosphorylation Gene Expression (mRNA) No rm al A er So why do we need to think about Proteomics? Gene expression Protein modifications Protein abundance 38
  • Proteomics: Key Technologies and Platforms Mass spectrometry - gets accurate mass of proteins, their peptide fragments upon digestion and their post-translational modifications Goal: to analyze complex sample of normal and diseased tissues to generate a protein “fingerprint”. Peptide sequencing (MS/MS) can tell you what protein is being affected. Tissue microarrays- This approach uses well-established immunohistochemistry techniques and formalin-fixed tissue samples Protein microarrays - Approach uses spotted proteins as bait for proteinprotein interaction studies 39
  • Proteomics using Mass Spectrometry From: Sebahat Ocak, et al. Proc Am Thorac Soc Vol 6. pp 159–170, 2009 40
  • Proteomics: A Clinical Pilot Study Need: new technologies for detecting early-stage ovarian cancer “Use of proteomic patterns in serum to identify ovarian cancer” by Petricoin et al (FDA/NIH/MD Anderson) - The Lancet 359, pg 572, 2002. Serum from 50 normal women Serum from 50 cancer patients Generated >15,000 protein mass spectra Identified 5-20 proteins that differ in normal patients and cancer patients (potential diagnostic markers) 41