Molecular Techniques Tools For Success - Hopkins Medicine

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Molecular Techniques:Tools for SuccessJason Rosenzweig, PhDFrom Bench to Bedside - Molecular Techniques: Tools for Success

Outline IntroductionDNA CloningPolymerase Chain Reaction (PCR)ElectrophoresisHybridizationArray-based expression profilingFrom Bench to Bedside - Molecular Techniques: Tools for Success

IntroductionFrom Bench to Bedside - Molecular Techniques: Tools for Success

DNAAlberts, et al. 2002From Bench to Bedside - Molecular Techniques: Tools for Success

RNAAlberts, et al. 2002From Bench to Bedside - Molecular Techniques: Tools for Success

ProteinsAlberts, et al. 2002From Bench to Bedside - Molecular Techniques: Tools for Success

DNA CloningFrom Bench to Bedside - Molecular Techniques: Tools for Success

DNA CloningDNA fragmentPlasmidDNA IsolationBacteriaCellCultureFrom Bench to Bedside - Molecular Techniques: Tools for Success

DNA Cloning - Vector ConstructionExpression Systems: Bacterial Yeast Baculovirus (Insect) Plant Mammalian Cell-freePromoterSelection marker/Resistance geneReplicationOriginFrom Bench to Bedside - Molecular Techniques: Tools for SuccessCloningsite

DNA Cloning - Restriction NucleasesType II Restriction nucleases: Recognize specific DNA sequences Usually four to eight bases and palindromic Cut the DNA – can be blunt or “sticky”Alberts, et al. 2002From Bench to Bedside - Molecular Techniques: Tools for Success

DNA Cloning - LigationSticky Ends Blunt Ends Alberts, et al. 2002From Bench to Bedside - Molecular Techniques: Tools for Success

Polymerase Chain ReactionFrom Bench to Bedside - Molecular Techniques: Tools for Success

Polymerase Chain ReactionDNA template and primersHeat to denature DNACool to anneal primersDNA polymerase uses original DNAas a template to make a copyResulting DNA is one strand of originaltemplate and one newly synthesized strandFrom Bench to Bedside - Molecular Techniques: Tools for Success

Quantitative PCR (qPCR)Dye-based (SYBR)DNA-binding dye fluoresces whenbound to double stranded DNAProbe-based (TaqMan)Probe labeled with a fluorophore and aquencher.Probe binds a specific sequence betweenthe primersProbe is digested during DNA synthesis,releasing fluorophore from quencher.PCRPCRFrom Bench to Bedside - Molecular Techniques: Tools for Success

qPCRFrom Bench to Bedside - Molecular Techniques: Tools for Success

Reverse Transcriptase PCR (RT-PCR)Quantifying gene expressionRNAReverse transcriptase, DNA primerscomplementary DNA (cDNA)RNAcDNA used as template for qPCRFrom Bench to Bedside - Molecular Techniques: Tools for Success

Gel ElectrophoresisFrom Bench to Bedside - Molecular Techniques: Tools for Success

Gel ElectrophoresisElectricity TimeFrom Bench to Bedside - Molecular Techniques: Tools for Success

Gel Electrophoresis – Two DimensionsIsoelectric Focusing648 6 57475 8548 6 6784 57Alberts, et al. 2002From Bench to Bedside - Molecular Techniques: Tools for Success

Hybridization TechniquesFrom Bench to Bedside - Molecular Techniques: Tools for Success

Hybridization Techniques Southern Blot (DNA) – labeled, single-strandednucleic acid probes to detect DNA Northern Blot (RNA) – labeled, single-strandednucleic acid probes to detect RNA Western Blot (Protein) – labeled probes to detectspecific protein (primary and secondary antibodies) Enzyme-Linked Immunosorbent Assay (ELISA) From Bench to Bedside - Molecular Techniques: Tools for Success

Hybridization – Southern and NorthernAlberts, et al. 2002From Bench to Bedside - Molecular Techniques: Tools for Success

Hybridization – Western BlotTransfer proteins from gel to membraneHybridizationVisualizationWinston et al. 2008From Bench to Bedside - Molecular Techniques: Tools for Success

Hybridization - ELISA Sensitive and specific Quantification ofpicogram levels of proteinde la Rica and Stevens, 2012From Bench to Bedside - Molecular Techniques: Tools for Success

Array-based assaysFrom Bench to Bedside - Molecular Techniques: Tools for Success

Array-based Expression ProfilingHybridization targets aremicroprinted on to glass slidesorchip-based arraysBlackburn and Shoko, 2011.From Bench to Bedside - Molecular Techniques: Tools for Success

Array-based Expression ProfilingRobinson et al., 2000From Bench to Bedside - Molecular Techniques: Tools for Success

Protein ArraysAnalytical: Antibody-based arrays forprotein detectionFunctional: Protein-based arrays fordetection of functional interactionsReverse Phase: Analyze many samples atonce.Sutandy et al. 2013From Bench to Bedside - Molecular Techniques: Tools for Success

From Bench to Bedside - Molecular Techniques: Tools for Success

References Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. NewYork: Garland Science; 2002.Armstrong, J. A. and Schulz, J. R. 2008. Agarose Gel Electrophoresis. CurrentProtocols Essential Laboratory Techniques. 00:7.2:7.2.1–7.2.20.Gallagher, S., Winston , S. E., Fuller, S. A. and Hurrell, J. G. 2008. Immunoblottingand Immunodetection. Current Protocols in Molecular Biology. 83:III:10.8:10.8.1–10.8.28.de la Rica, R. and Stevens, M. M. 2012. Nature Nanotechnology 7, 821–824.Robinson, B.W.S., Erle, D.J., Jones, D.A., Shapiro, S. Metzger, W.J., Albelda, S.M.,Parks, W.C., Boylang A. 2000. Recent advances in molecular biological techniquesand their relevance to pulmonary research. Thorax 55:329-339Blackburn, J.M. and Shoko, A. Protein Microarrays: Methods and Protocols.Methods in Molecular Biology. 785:305-330.Sutandy, F. R., Qian, J., Chen, C.-S. and Zhu, H. 2013. Overview of ProteinMicroarrays. Current Protocols in Protein Science. 72:27.1:27.1.1–27.1.16.From Bench to Bedside - Molecular Techniques: Tools for Success

Analytical: Antibody-based arrays for protein detection Functional: Protein-based arrays for detection of functional interactions . From Bench to Bedside - Molecular Techniques: Tools for Success References Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Armstrong, J .

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