Instrumentation EOS PDF

ELECTROPHORESIS 1 By Dr Tsen Min Tze Inspire Empower Elevate  Learning Outcomes Principles of electrophoresis Agarose gel electrophoresis (DNA) Apparatus for agarose gel electrophoresis Capillary electrophoresis Electrophoresis Electrophoresis is a tech...

ELECTROPHORESIS 1 By Dr Tsen Min Tze Inspire Empower Elevate  Learning Outcomes Principles of electrophoresis Agarose gel electrophoresis (DNA) Apparatus for agarose gel electrophoresis Capillary electrophoresis Electrophoresis Electrophoresis is a technique used to separate and sometimes purify charged macromolecules that differ in size, charge and conformation in an electric field. When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge.  The greater the charge of a macromolecule, the faster it migrates (greater electrophoretic mobility). Factors Affecting Electrophoresis Greater size of the macromolecule will have greater frictional and electrostatic forces, slower electrophoretic mobility. Rounded molecules have lesser frictional and electrostatic retardation compared to non- globular structure. Globular protein move faster than fibrous protein; supercoil plasmid move faster than linear plasmid. Supercoiled plasmid migrate faster than linearized plasmid. Electrophoresis - Principles Proteins and nucleic acids migrate within a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. These are solid yet porous matrixes. molecules will move through the matrix at different rates, usually determined by their mass, charge and conformation; they eventually get separated as different ‘bands’. Molecular weight markers are run side by side with samples, the molecular weight of samples can be estimated by referring to the markers. Electrophoresis - Commonly Used Matrixes DNA electrophoresis - Agarose gel. Protein electrophoresis - Polyacrylamide gel.  Agarose Gel Electrophoresis (DNA) Agarose gel is used to separate DNA fragments based on molecular weight. DNA (and RNA) molecules are negatively charged, when placed in an electric field, they migrate to the positively charged anode. DNA fragments (double-stranded linear DNA) has a uniform mass/charge ratio; thus DNA fragments are separated by size within an agarose gel. Shorter fragments migrate faster, longer fragment migrate slower. The distance migrated on gel correlate inversely with the logarithm of molecular weight. How to cast an agarose gel? Gel tray Boil agarose powder in buffer Wait for the gel to set Insert a comb  Loading DNA into wells 1475-9292-5-2-1 Loading Dye While loading the DNA sample into the gel, DNA sample is mixed with loading dye. Loading dye typically contains glycerol (or sucrose) and Bromophenol Blue. Glycerol –high density solution– pull DNA sample and settle to the bottom of the well. Bromophenol Blue – small dye molecules that move through the gel rapidly ahead of all DNA fragments. It is therefore acting as a marker for the electrophoretic front. (Electrophoresis will be stopped before all DNA samples migrate and run out from the end of the gel.) Loading Dye Sample is loaded here. Direction of DNA migration Loading dye pull DNA samples to the bottom of Electrophoretic front the well. DNA Ladder AKA DNA molecular weight marker. It is a set of DNA standards with known base pair size that are used to identify the approximate size of a DNA sample run on electrophoresis. DNA ladder is run side by side with DNA samples. Staining DNA in Agarose Gel DNA are invisible to naked eyes. Ethidium bromide bind to double stranded DNA. Ethidium bromide fluoresce in orange color while irradiated by UV light. Ethidium bromide is normally added when the gel is made; or added to the electrophoresis buffer. After electrophoresis, the gel will be irradiated with UV, image will be taken. Safer alternative: crystal violet, methylene blue, propidium iodate … Staining DNA in Agarose Gel Ethidium bormide bind to Gel image taken and turn double stranded DNA. into black and white image. Ethidium bromide fluoresce when the gel is irradiated with UV light. Capillary Electrophoresis (I hate definition) A method to separate charged molecules by their charge to mass ratio in a capillary tube using electric field and electroosmotic flow. It combines the features of electrophoresis (move charged molecules in an electric field) and chromatography (measure the time required for molecules to travel through a matrix). Automated DNA sequencer. Schematic of capillary electrophoresis system  Electroosmotic Flow Electroosmotic flow (EOF) is the force that moves fluid from one end of a capillary tube to another. The concept of EOF is extremely complex; fortunately, it is possible to carry out capillary electrophoresis without understanding the theory behind it. Electroosmotic Flow Electroosmotic Flow  The inner surface is coated with negatively charged groups. Capillary is filled with buffer and samples. Negative groups attract cations to the inside wall of the capillary, forming a cations double layers: rigid layer and diffuse layer. Diffuse layer is rich in positive charge, but is mobile, not rigidly bound to capillary wall. When a high voltage is applied through the capillary, the cations in the diffuse layer are attracted to the negative electrode, dragging the bulk buffer solution with them (electroosmotic flow). The net result is that this electroosmotic flow will move everything to the negative electrode (cathode). Cations Electrophoretic Mobility + EOF  Fastest Neutral EOF only (not attracted to any electrode) Anions Opposite Electrophoretic Mobility + EOF  Slowest Electroosmotic Flow + When running voltage is too high in electrophoresis, DNA band with turn into parabolic line; due to friction. If the DNA band is driven by electroosmotic pressure, no friction between the band and the capillary tube. The band is uniform across the capillary. - Smiley gel – when voltage applied during electrophoresis is too high. Capillary Electrophoresis – Advantages over Conventional Electrophoresis 1. High speed – because high voltage can be used. 2. High resolution – able to resolve DNA fragments with only 1- bp difference. 3. Yield precise quantitative information – detector to detect the migrated DNA, convert by software into quantitative information. 4. Use only very little sample. THANK YOU ELECTROPHORESIS 2 By Dr Tsen Min Tze  Learning Outcomes SDS-PAGE (protein) Native PAGE vs denaturing PAGE Isoelectric focussing (IEF) and 2D-gel electrophoresis Western blot ELISA SDS-PAGE Sodium dodecyl sulfate – polyacrylamide gel electrophoresis Resolve and separate proteins based on molecular weight SDS-PAGE - Principles Proteins are first mixed (and heated in) Laemmli buffer before electrophoresis. Laemmli buffer contains: 1. SDS – An anionic detergent which denatures secondary structures, and gives an overall negative charge to each protein in proportion to its mass. 2. 2-mercaptoethanol - Breaks disulfide bonds in globular protein, and hence disrupt protein tertiary and quaternary structure. 3. Glycerol – High density solution to pull protein sample down and stay in it designated well. 4. Dye – Usually bromophenol blue. Action of SDS and 2-mercaptoethanol on proteins. Protein separation by SDS-PAGE  SDS-PAGE - Principles Because of the action of SDS and 2-mercaptoethanol, all denatured proteins will therefore become peptide chains with fairly constant charge to mass ratios. The electrophoretic migration rate through a gel is therefore determined only by the size of the protein. All proteins, because of the negative charge imparted by SDS, move to positive electrode. Molecular weights are determined by simultaneously running marker proteins of known molecular weight. After electrophoresis, gels are stained with Coomassie Blue or Silver stain to visualise band patterns.  Native vs. Denaturing PAGE In native PAGE, proteins are not denatured, ie no SDS or 2-ME are added before electrophoresis. Proteins migrate through PAGE gel according to their charge, mass and shape (conformation). After electrophoresis, proteins remain in their native conformation, they can be isolated from gel for use in other assays. Two Dimensional Gel Electrophoresis Isoelectric point (pI) - the pH at which a particular molecule or surface carries no net electrical charge. Proteins are composed of amino acids. Amino acids can be neutral, positive or negatively charged. Hence a protein molecule has an overall net charge. This net charge is pH-dependant. Protein can become more positive or negatively charged due to the loss or gain of protons (H+). Have positive charge in buffer with pH below the pI. Have negative charge in buffer with pH above the pI. Have neutral charge in buffer with pH=pI. Two Dimensional Gel Electrophoresis Isoelectric focussing (IEF) – an electrophoretic separation technique based on the isoelectric points of molecules. In contrast to other electrohoretic technique, pH is not kept constant throughout the whole system. Instead, a pH gradient is set up in a gel strip. Proteins are loaded, they will move to anode/cathode according to their net charge. They will reach a point where the gel pH is equal to their pI. Because proteins have zero net charge when pH = pI, they cease moving. The protein is said to ‘focus’ at this point. pH gradient gel strip Loading protein sample Electrophoresis  Two Dimensional Gel Electrophoresis An electrophoretic separation technique to separate protein mixtures by two different principles in order to improve resolution. Proteins are first separated by isoelectric focussing using a pH gradient strip. The pH gradient strip is then treated with buffer with SDS and reducing agent. Proteins are then further separated SDS-PAGE in a perpendicular direction. After completion of 2-D electrophoresis, gel will be stained with silver stain or Coomassie Blue. A 2-D gel after staining with Coomassie Blue or silver staining.  Proteins are separated first by isoelectric focussing using a pH gradient gel stip. The gel strip is then transferred onto the surface of a vertical SDS-PAGE gel. Proteins are then further separated by SDS – PAGE by their size in a perpendicular direction. Loading a gel strip onto the surface of a vertical SDS-PAGE gel Western Blot Western blot is a laboratory method used to separate and detect specific protein molecules from a mixture of proteins. The main steps are: 1. Sample preparation and electrophoresis 2. Electrophoretic transfer 3. Blocking 4. Antibody incubation and detection Western Blot Sample Preparation and Electrophoresis Total protein is extracted from cells. Protein mixture is heated with loading buffer to denature higher order structure; and to impart negative charge to the proteins. Protein mixture will be then separated by size using SDS-PAGE. Electrophoretic Transfer AKA blotting. Transfer of all protein bands on polyacrylamide gel to a membrane using an electric field. Nitrocellulose or PVDF (Polyvinylidene fluoride) membrane. Close contact of gel and membrane to ensure effective transfer. Membrane must be placed in the positive electrode, so negatively charged proteins will move from gel to the membrane when voltage is applied. Electrophoretic Transfer Transfer sandwich Transfer tank with transfer buffer Blocking Blocking is to prevent antibodies from binding the membrane surface nonspecifically. Membrane is immersed in bovine serum albumin (BSA) or skim milk solution. Blocking Antibody incubation and detection Staining with primary and secondary antibodies. Primary antibodies bind specifically to the target protein. Secondary antibodies are conjugated with enzyme. Then add substrate solution. Enzyme will react with substrate to give colored product. The color will be visible only in the target protein band. There are many detection methods… Example results…. ELISA Enzyme-linked Immunosorbent Assay  ELISA An immunoassay in which small molecules like antigen, antibodies, peptides or proteins can be detected and quantified. 1. Simple and sensitive. 2. Quantitative – Intensity of signal gives quantitative information. 3. Versatile – many ready-to-use detecting formats: enzyme-conjugated antibodies or fluorescence-conjugated antibodies. ELISA – Main Reagents Antigen (the target protein) Antibody Enzyme Conjugated Antibody Substrate ELISA – Direct ELISA ELISA – Indirect ELISA * Washing to remove unbound antibodies THANK YOU Polymerase chain reaction and thermal cycler I Yih-Yih Kok [adapted from Dr Wong Ying Pei’s note] Outlines Conventional Linear Gradient Techniques, instrumentation and detectors Application Copyright reserved © 2020, IMU. All rights reserved  Polymerase chain reaction (PCR) Amplification of a small amount target DNA sequence on a DNA template into large quantities of DNA sequences by thermal cycler 1 copy  millions of copies in 3 hrs (20 2n) Generate sufficient copies of specific DNA sequence for : Diagnosis Therapeutic development Genotyping Sequencing Forensic analysis Copyright reserved © 2020, IMU. All rights reserved https://bio1151.nicerweb.com/Locked/media/ch14/genes.html  Types of PCR Polymerase chain reaction Conventional Real time Digital PCR PCR PCR https://www.bio-rad.com/en-sg/category/thermal-cyclers-for-pcr?ID=75f1b406-3746-4580-a998-74245b094f56 http://www.tecnologie-salute.unibo.it/risorse-strumentali/epigenlab/real-time-pcr-detection-system-iq5-biorad Copyright reserved © 2020, IMU. All rights reserved https://www.bioradiations.com/qx100-droplet-digital-pcr-system/  Thermal cycler Laboratory instrument use to amplify target sequence of DNA via the PCR process https://www.biocompare.com/Nucleic-Acid-Electrophoresis/23396-Thermal-Cyclers-Thermocyclers-96-Well/?vendor=106079&vids=100041 Copyright reserved © 2020, IMU. All rights reserved https://www.marshallscientific.com/Eppendorf-5333-MasterCycler-Thermal-Cycler-p/ep-mc5333.htm Anatomy of Thermal cycler Thermal block Hot Bonnet Air Exhaust Lid vents Control Panel Air Intake vents Thermal cycler raises and lowers the temperature of the block in discrete, pre-programmed steps. Copyright reserved © 2020, IMU. All rights reserved  PCR 1 copy  millions of copies (20 2n) Copyright reserved © 2020, IMU. All rights reserved https://www.bosterbio.com/protocol-and-troubleshooting/pcr-principle  Chemicals required for PCR Components Functions Template DNA isolated from the source. Contains the region of the DNA fragment to be amplified Primers Short oligonucleotides designed to recognise the region on (forward and reverse) DNA Complementary to the DNA regions at the 5' and 3' ends of the DNA region deoxynucleotide Free dNTPs added during the elongation process to form triphosphates double stranded DNA (dNTPs) Typical concentration of each dNTP = 200 µM DNA polymerase Enzymes that catalysing the elongation process to synthesis DNA chain PCR reaction volume = 20 – 50 µL Taq DNA polymerase PCR cycles = 30 – 40 cycles PCR buffers Provides a suitable chemical environment for optimum activity and stability of the DNA polymerase Reaction tubes = 200 µL or 500 µL Some consists of divalent ions (such as Mg2+) stabilises the reaction Nuclease Free water Prevent nuclease contamination in PCR reaction Maintaining the final volume Copyright reserved © 2020, IMU. All rights reserved PCR https://www.youtube.com/watch?v=iQsu3Kz9NYo Copyright reserved © 2020, IMU. All rights reserved  Polymerase chain reaction (PCR) : double stranded DNA Denaturation Form 2 single stranded DNA Heating at 90 – 100 °C, 1 – 2 minutes https://www.biosyn.com/faq/What-is-needed-to-amplify-a-segment-of-DNA.aspx Copyright reserved © 2020, IMU. All rights reserved  Polymerase chain reaction (PCR) Annealing Primers bind to complementary sequence before forming new strand DNA 50 – 70 °C, 15 – 60s Annealing temperature based on the melting temperature (Tm) of primers Optimisation is required: Non specific Primers amplification 18 – 30 bp bind complementary to DNA Primer-dimer GC content = 40 – 60% formation Copyright reserved © 2020, IMU. All rights reserved https://www.biosyn.com/faq/What-is-needed-to-amplify-a-segment-of-DNA.aspx  Polymerase chain reaction (PCR) Extension/Elongation DNA polymerase attaches to the primers Elongate the new DNA strands by adding in dNTPs 60 – 75 °C, 15 – 60s Forms 2 news DNA sequences (20 2n) Next cycles Copyright reserved © 2020, IMU. All rights reserved https://www.biosyn.com/faq/What-is-needed-to-amplify-a-segment-of-DNA.aspx  Polymerase chain reaction (PCR) Copyright reserved © 2020, IMU. All rights reserved https://www.bosterbio.com/protocol-and-troubleshooting/pcr-protocol  Conventional PCR End point PCR Qualitative results Agarose gel electrophoresis analysis https://www.bosterbio.com/protocol-and-troubleshooting/pcr-principle Copyright reserved © 2020, IMU. All rights reserved Khalafalla AI, Al-busada KA, El-Sabagh IM.. Multiplex PCR for rapid diagnosis and differentiation of pox and pox-like diseases in dromedary Camels. Virology Journal 2015.  Activity Setting up Conventional PCR programme IF a basic cycle sequencing protocol consisting of 30 repeats of denaturation, annealing and extension step. Rather than listing all 90 steps, design a short, easy to enter program: https://www.eppendorf.com/product-media/doc/en/45949/Eppendorf_PCR_Brochure_Mastercycler-Family_nexus_pro_Amplify.pdf Copyright reserved © 2020, IMU. All rights reserved  Activity Setting up Conventional PCR programme https://www.thermofisher.com/my/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-pcr/thermo-scientific-pcr-enzymes-master-mixes/hot-start-dna- Copyright reserved © 2020, IMU. All rights reserved polymerases-master-mixes-thermo-scientific/dreamtaq-dna-polymerase.html  Gradient PCR Enables rapid testing of the optimum annealing temperature of a particular primer A temperature gradient, can be programmed between 1 oC - 20 oC, is built up across the thermo-block. Analyses several primers simultaneously in a single PCR profile. Copyright reserved © 2020, IMU. All rights reserved http://www.techne.com/adminimages/A01_001B_PCR_Optimisation_gradient.pdf  Activity Setting up Gradient PCR programme If a basic cycle sequencing protocol consisting of 30 repeats of denaturation, annealing and extension step. And, few primers were used. Rather than listing all 90 steps, design a short, easy to enter program: 60-65 60/65 60-65 60-65 Copyright reserved © 2020, IMU. All rights reserved https://www.labnetinternational.com/sites/www.labnetinternational.com/files/product-images/TC020_Mullti_Gene_Mini_0.png  Reverse transcription PCR (RT-PCR) mRNA cDNA Reverse Transcription Denaturation Annealing Extension Copyright reserved © 2020, IMU. All rights reserved https://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/molecular-biology/pcr/how_reverse_transcriptase%20(RT)%20PCR%20works_big.png N PCR vs RT-PCR PCR RT-PCR PCR RT-PCR Copyright reserved © 2020, IMU. All rights reserved One step vs Two steps (RT-PCR) One-step RT PCR All reactions occurs in one tube (synthesis of cDNA +PCR) Two-step RT PCR Synthesis cDNA first, then only PCR Occurs in different tubes Copyright reserved © 2020, IMU. All rights reserved  Activity Knowledge check List TWO (2) differences between PCR and RT-PCR. Copyright reserved © 2020, IMU. All rights reserved PCR vs RT-PCR PCR RT-PCR PCR RT-PCR Copyright reserved © 2020, IMU. All rights reserved  Activity Knowledge check List FIVE (5) applications of PCR Copyright reserved © 2020, IMU. All rights reserved  Activity Knowledge check List FIVE (5) applications of PCR Molecular diagnosis Sequencing Genetic testing Drug discovery Phylogenetics Mutation screening Genetic cloning Gene expression studies Copyright reserved © 2020, IMU. All rights reserved References and Extra resources Garibyan L, Avashia N. Polymerase chain reaction. J Invest Dermatol. 2013;133(3):1-4. Valones MA, Guimarães RL, Brandão LA, de Souza PR, de Albuquerque Tavares Carvalho A, Crovela S. Principles and applications of polymerase chain reaction in medical diagnostic fields: a review. Braz J Microbiol. 2009;40(1):1-11 Wang X, Seo DJ, Lee MH, Choi C. Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species. Journal of Clinical Microbiology 2014; 52 (2): 557-563 Ishmael FT, Stellato C. Principles and applications of polymerase chain reaction: basic science for the practicing physician. Ann Allergy Asthma Immunol. 2008; 101(4):437-43 Petz LN, Turell MJ, Padilla S, et al. Development of conventional and real-time reverse transcription polymerase chain reaction assays to detect Tembusu virus in Culex tarsalis mosquitoes. Am J Trop Med Hyg. 2014;91(4):666-671 Copyright reserved © 2020, IMU. All rights reserved THANK YOU Polymerase chain reaction and thermal cycler II Yih-Yih Kok [adapted from Dr Wong Ying Pei’s note] Outline Real-time PCR Detection chemistry Analytical software Techniques, instrumentation and detectors Application Copyright reserved © 2020, IMU. All rights reserved Activity Basic principle of PCR process 20 2n Copyright reserved © 2020, IMU. All rights reserved  Real time PCR aka Quantitative PCR (qPCR) Allows monitoring the newly generated PCR product during the PCR run with the help of fluorescence labeled oligonucleotide Determine HOW gene expression changes over time using amplification curve Determine WHEN does signal generation and involved determination of the cycle threshold (Ct) value. [DNA/ RNA] , Ct value Fluorescent Signal ∝ Number of Amplicons Copyright reserved © 2020, IMU. All rights reserved https://www.bio-rad.com/en-sg/applications-technologies/what-real-time-pcr-qpcr?ID=LUSO4W8UU What is PCR and qPCR? https://www.youtube.com/watch?v=rpLSvEbOmqc Copyright reserved © 2020, IMU. All rights reserved https://geneticeducation.co.in/differences-between-pcr-vs-qpcr/#google_vignette Advantages of qPCR Rapid Highly Highly detection specific sensitive detection detection Reduced risk Automation of false and high Quantification positive throughput Copyright reserved © 2020, IMU. All rights reserved Applications of qPCR Gene expression profiling miRNA expression profiling analysis Diagnosis (pathogen detection/ viral quantification) Copy numbers detection/ variant analysis SNP genotyping and allelic discrimination Somatic mutation studies Chromatin IP quantification Copyright reserved © 2020, IMU. All rights reserved Methodology qPCR Copyright reserved © 2020, IMU. All rights reserved Overview of qPCR https://www.youtube.com/watch?v=1kvy17ugI4w Copyright reserved © 2020, IMU. All rights reserved  RFU Activity Cycle Threshold (Ct)- Number of cycle required for fluorescent signal to cross the threshold Threshold line Ct https://www.biocompare.com/Bench-Tips/343854-qPCR-Checklist-Steps-to-Better-Results/ Copyright reserved © 2020, IMU. All rights reserved  qPCR vs RT-qPCR Fluorescent dye https://www.biocompare.com/Bench-Tips/343854-qPCR-Checklist-Steps-to-Better-Results/ Copyright reserved © 2020, IMU. All rights reserved  Detection chemistry- sequence-unspecific detection using intercalating fluorescent dye binding to the minor groove of the amplified DNA sequences leads to fluorescence emission SYBR® Green I, DYBR ® Gold, BEBO, BOXTO, EvaGreen, YO-PRO-1 Copyright reserved © 2020, IMU. All rights reserved https://www.sigmaaldrich.com/technical-documents/protocols/biology/sybr-green-qpcr.html  Detection chemistry-Sequence specific probes detect specific PCR products via employing Dual labeled probes, Taqman® fluorophore-linked oligonucleotides small fluorescent molecules attached to oligonucleotides Primer-probes, probes, analogues of nucleic acids donor (reporter) and acceptor (quencher)  FRET Molecular Beacons Scorpions Uni-probe Hybridisation probe https://www.sigmaaldrich.com/technical-documents/articles/biology/scorpions-probes.html https://www.sigmaaldrich.com/technical-documents/articles/biology/molecular-beacons.html https://www.sigmaaldrich.com/technical-documents/articles/biology/dual-labeled-probes.html Copyright reserved © 2020, IMU. All rights reserved https://www.sigmaaldrich.com/technical-documents/articles/biology/lightcycler-probes.html  The fluorescent reporter emits its energy after being released and can be quantified using a computer https://www.researchgate.net/figure/Comparison-of-intercalating-dye-and-hydrolysis-based-probe-detection-A-SYBR-R-Green_fig3_342182497 Sequence-unspecific detection Advantages Disadvantages Fluorescent intensity proportional No target specificity of the dye to the amount of PCR products Possibility of generation of false Possibility of monitoring the positive signals amplification of any dsDNA Recognition of products require No requirement for specific probe melting curve analysis design Decreased assay set-up time and cost Copyright reserved © 2020, IMU. All rights reserved  Dyes used in qPCR Copyright reserved © 2020, IMU. All rights reserved https://www.qiagen.com/fi/service-and-support/learning-hub/molecular-biology-methods/pcr/#PCR%20primer%20design  Real-time PCR – Detection chemistry Light sources  LED, halogens lamps, Laser Detector  photodiode, CCD, photomultiplier tube Copyright reserved © 2020, IMU. All rights reserved https://www.bio-rad.com/en-sg/applications-technologies/introduction-qpcr-instrumentation?ID=LUSO5YMNI#1  Data Analysis NC= negative control PC= Positive control Analysis based on Threshold line control / standard curve  absolute quantification or relative quantification Amplification curve  Cycle, relative fluorescence units  Ct value Melting curve  derivative fluorescence, temperature  primer dimer Copyright reserved © 2020, IMU. All rights reserved © Mousavi et al. J Bacteriol Parasitol 2018, 9:4  Data Analysis Absolute quantification Relative quantification serially diluted standards of known Gene expression levels are calculated by the ratio concentrations to generate a standard curve. between the amount of target gene and an linear relationship between Ct and initial endogenous reference gene. amounts of template, concentration of unknowns are determined based on their Ct values Delta (∆)∆Ct method = ∆Ctsample – ∆Ctcontrol Fold change = 2- ∆∆Ct https://figshare.com/articles/_Determination_of_the_detection_limit_of_the_real_time_PCR_and_standard_curve_/883372 Copyright reserved © 2020, IMU. All rights reserved  Data Analysis Amplification efficiency Amplification efficiency (E) = 10(-1/slope) % E = [-1+ 10(-1/slope)] x 100 % Ratio of the number of target gene molecules at the end of a PCR cycle divided by the target molecules at the start of the same PCR cycle. desired amplification efficiencies range from 90% to 110% Ren X, Yue X, Mwakinyali SE, Zhang W, Zhang Q, Li P. Small Molecular Contaminant and Microorganism Can Be Simultaneously Detected Based on Nanobody- Copyright reserved © 2020, IMU. All rights reserved Phage: Using Carcinogen Aflatoxin and Its Main Fungal Aspergillus Section Flavi spp. in Stored Maize for Demonstration. Front. Microbiol. 2020; 10:3023  Activity Quantify the expression of TNF- alpha TNF alpha Ct value GAPDH Ct value Treated cells Untreated cells Copyright reserved © 2020, IMU. All rights reserved Criteria for a perfect PCR system Components Descriptions Template Good quality DNA or RNA template Concentration No contamination Primers Design the appropriate primers Optimum annealing temperature Detection qPCR or end point analysis system PCR cycling Correct PCR cycling Copyright reserved © 2020, IMU. All rights reserved POP QUIZ PCR and qPCR What are the differences between PCR and qPCR? Copyright reserved © 2020, IMU. All rights reserved https://geneticeducation.co.in/differences-between-pcr-vs-qpcr/#google_vignette POP QUIZ PCR and qPCR How does qPCR works? Copyright reserved © 2020, IMU. All rights reserved https://www.researchgate.net/figure/Comparison-of-intercalating-dye-and-hydrolysis-based-probe-detection-A-SYBR-R-Green_fig3_342182497 POP QUIZ PCR and qPCR What are the PCR inhibitors may affect the PCR result? Copyright reserved © 2020, IMU. All rights reserved POP QUIZ PCR and qPCR Name 2 applications of qPCR in: Diagnosis SNP analysis Gene expression Copyright reserved © 2020, IMU. All rights reserved References and Extra resources Seifi M, Ghasemi A, Heidarzadeh D, Khosravi M, Namipashaki A, Soofiany VM, Khosroshahi AA, Danaei N. Overview of real-time PCR principle 2012. Navarro E, Serrano-Heras G, Castaño MJ, Solera J. Real-time PCR detection chemistry. Clin Chim Acta. 2015; 439:231-50 Stephen A Bustin, Vladimir Benes, Jeremy A Garson, Jan Hellemans, Jim Huggett, Mikael Kubista, Reinhold Mueller, Tania Nolan, Michael W Pfaffl, Gregory L Shipley, Jo Vandesompele, Carl T Wittwer, The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clinical Chemistry 2009; 55 (4): 611–622 Ganger, MT, Dietz GD, Ewing SJ. A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments. BMC Bioinformatics 2017; 18: 534 https://www.qiagen.com/fi/service-and-support/learning-hub/molecular-biology-methods/pcr/ Ruijter JM, Ramakers C, Hoogaars WM, et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 2009;37(6):e45 Copyright reserved © 2020, IMU. All rights reserved THANK YOU  DNA SEQUENCER AND PROTEIN SEQUENCER LEARNING OBJECTIVES To understand: a. Chemistry b. Principle and methods of sequencing c. Technique and instrumentation d. Application DNA SEQUENCING  INTRODUCTION “Sequencing” means finding the order of nucleotides on a piece of DNA Nucleotide order determines amino acid order, and by extension, protein structure and function (proteomics) An alteration in a DNA sequence can lead to an altered or non functional protein, and hence to a harmful effect in a plant or animal. Understanding a particular DNA sequence can shed light on a genetic condition and offer hope for the eventual development of treatment. DNA technology is also extended to environmental, agricultural and forensic applications DNA SEQUENCING Process of determining the precise order of nucleotides within a DNA molecule Methods:  Maxam-Gilbert sequencing  Sanger sequencing (chain-termination method, dideoxy method)  Automated sequencing  Shotgun sequencing  Pyrosequencing  DNA Sequencing Methods In 1977 two separate methods for sequencing DNA were developed: Maxam & Gilbert sequencing or Chemical degradation method, using chemical sequencing Chain termination method or cycle sequencing or Sanger method, using dideoxynucleotides Modern sequencing equipment uses the principles of the Sanger technique.  MAXAM-GILBERT SEQUENCING Based on chemical modification of DNA and subsequent cleavage at specific bases 1) Radioactive labeling at 5' end of the DNA 2) Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions Dimethyl Formic Hydrazi H+ sulfate acid ne (H) NaCl 3) Cleaved by piperidine  MAXAM-GILBERT SEQUENCING 4) Gel electrophoresis & autoradiography Longer Reading Shorter MAXAM-GILBERT SEQUENCING Limitations: Requires extensive use of hazardous chemicals  Use of radioactivity and toxic chemicals Relatively complex set-up/technical complexity Difficult to "scale-up“  Cannot be used to analyze more than 500 base pairs  Gel electrophoresis is limited to 700-900 bp, with 400-500 bp more commonly attained  The first 15-40 bp are often difficult to interpret Read-length decreases from incomplete cleavage reactions Difficult to make Maxam-Gilbert sequencing based DNA kits Not widely used SANGER SEQUENCING Based on the selective incorporation of chain-terminating dideoxynucleotides (ddNTP) by DNA polymerase during in vitro DNA replication ddNTPs are essentially the same as nucleotides except they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH)  SANGER SEQUENCING 1) The DNA to be sequenced is prepared as a single strand 2) This template DNA is supplied with a mixture of all four normal (deoxy) nucleotides in ample quantities 3) Add DNA polymerase and primers (radiolabeled with 32P) 4) Add to 4 reaction mixtures consisting the different dideoxynucleotides (ddNTP) * The ddNTPs present in limiting quantities  SANGER SEQUENCING As the DNA is synthesized, dNTPs are added on to the growing For example if we looked at only the "G" tube, we might chain by the DNA find a mixture of the following: polymerase However, on occasion a ddNTP is incorporated into the chain in place of a normal nucleotide, which results in a chain- terminating event The result is a series of fragments of different lengths in each tube SANGER SEQUENCING 5) All reaction mixtures are electrophoresed on acrylamide gel under denaturing condition – DNA becomes single strand * Shorter strands move to the bottom of gel more quickly than the longer strands 6) DNA is autoradiographed for visualization – appear as horizontal bands on X-ray film AUTOMATED SEQUENCING Innovation of dideoxy sequencing Instead of having 4 separate tubes, this method only involves one tube containing all four ddNTPs Each ddNTP has different-colored flourescent label attachment Incubate the target DNA with dNTPs and DNA pol. along with ddNTPs and the sample is then loaded into single lane of gel The resulting bands are detected via laser and florescence detector – record amount of fluorescent emission Electropherogram DNA Sequencer ABI 3100 DNA SEQUENCER Each sample tray has 96 wells (1 per sample), and the analyzer (3100 model) has the capacity to analyze 16 wells at a time Robotic apparatus moves the sample tray so each of the 16 wells is in contact with a separate capillary tube filled with a polymer - this replaces a lane on an electrophoresis gel Labeled DNA from that well moves up the capillary tube, with smaller labeled fragments moving more quickly than longer ones DNA SEQUENCER A laser ‘reads’ the fluorescent label on each fragment as it passes up the capillary tube It takes 4 hours to ‘run’ 16 samples. The robotics then move the capillaries through a cleaning phase and move the tray of samples so the next 16 samples are processed. It takes 24 hours to process 96 samples. Electronic signals from the laser go to a sequencer programme and are converted into an electronic file of the code  Used for sequencing long DNA SHOTGUN SEQUENCING strands or the whole genome DNA is broken up randomly into numerous small segments and overlapping regions are identified between all the individual sequences that are generated Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence  Sequencing primer is hybridized to a single stranded, PCR amplified, DNA template and incubated with enzymes PYROSEQUENCING The first of four dNTPs is added to the reaction DNA polymerase catalyzes the incorporation of the dNTP into the DNA strand Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide Sulfurylase quantitatively converts PPi to ATP This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP The light produced in the luciferase-catalyzed reaction is detected and seen as a peak in a pyrogram Each light signal is proportional to the number of nucleotides incorporated Apyrase, a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP When degradation is complete, another dNTP is added As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peak in the pyrogram PYROSEQUENCING Pyrogram DE NOVO SEQUENCING De novo sequencing refers to sequencing a novel genome where there is no reference sequence available for alignment. Sequence reads are assembled as contig - a series of overlapping DNA sequences used to make a physical maps The coverage quality of de novo sequence data depends on the size and continuity of the contigs (ie, the number of gaps in the data). DE NOVO SEQUENCING The initial generation of the primary genetic sequence of a particular organism is called de novo sequencing. A detailed genetic analysis of any organism is possible only after de novo sequencing has been performed. De novo sequencing is typically accomplished by assembling individual sequence reads into longer contiguous sequences (contigs) or correctly ordered contigs (scaffolds) in the absence of a reference sequence. APPLICATION De novo sequencing generates an initial genomic sequence of a particular organism without a reference genome. Can be applied to research of animals, plants, and microorganisms, including phylogenetic studies, analysis of species diversity, genetic markers, and other genomic research. ADVANTAGES OF DE NOVO SEQUENCING Generates accurate reference sequences, even for complex or polyploid genomes Provides useful information for mapping genomes of novel organisms or finishing genomes of known organisms Clarifies highly similar or repetitive regions for accurate de novo assembly Identifies structural variants and complex rearrangements, such as deletions, inversions, or translocations METHODS FOR DE NOVO SEQUENCING Historically, de novo sequencing was carried out using capillary electrophoresis (CE) sequencers. With its long read lengths and high accuracy, CE-based sequencing made overlap consensus assembly the gold-standard technology for de novo projects. However, more recently, the high-throughput capabilities of massively parallel sequencing and the development of short-read assemblers have significantly reduced the time and cost associated with sequencing an entire genome. Next Generation Sequencing  454 Genome Sequencer  Illumina (Solexa) sequencing  SOLiD sequencing  SMRT sequencing  Complete Genomics sequencing PROTEIN SEQUENCING PROTEIN SEQUENCING Protein sequencing is determining the amino acid sequences of its constituent peptides; and also determining what conformation it adopts and whether it is complexed with any non-peptide molecules. The first protein sequencing was achieved by Frederic Sanger in 1953. He determined the amino acid sequence of bovine insulin. Sanger was awarded the Nobel Prize in 1958. APPLICATIONS 1) Identification of the protein family to which a particular protein belongs and finding the evolutionary history of that protein. 2) Prediction of the sequence of the gene encoding the particular protein. 3) Discovering the structure and function of a protein through various computational methods and experimental methods. 4) New protein biomarkers can be identified. 5) New drug targets can be identified. 6) Protein engineering studies. PROTEIN SEQUENCING METHODS Direct Methods: Edman degradation Mass spectrometry Indirect Methods: Amino acid sequence from the DNA or mRNA sequence encoding the protein EDMAN DEGRADATION The sequence of amino acids in a protein or peptide can be identified by Edman degradation, which was developed by Pehr Edman. This method can label and cleave the peptide from N-terminal without disrupting the peptide bonds between other amino acid residues. The Edman degradation reaction was automated in 1967 by Edman and Beggs. Nowadays, the automated Edman degradation (the protein sequenator) is used widely, and it can sequence peptides up to 50 amino acids. PROTEIN STRUCTURE  EDMAN DEGRADATION Cyclic degradation of peptides based on the reaction of phenylisothiocyanate with the free amino group of the N-terminal residue such that amino acids are removed one at a time and identified as their phenylthiohydantoin derivatives. An uncharged peptide is reacted with phenylisothiocyanate (PITC) at the amino terminus under mildly alkaline conditions to give a phenylthiocarbamoyl derivative (PTC-peptide). Then, under acidic conditions, the thiocarbonyl sulfur of the derivative attacks the carbonyl carbon of the N-terminal amino acid. The first amino acid is cleaved as anilinothiazolinone derivative (ATZ-amino acid) and the remainder of the peptide can be isolated and subjected to the next degradation cycle. Once formed, this thiazolone derivative is more stable than phenylthiocarbamyl derivative. The ATZ amino acid is then removed by extraction with ethyl acetate and converted to a phenylthiohydantoin derivative (PTH-amino acid). EDMAN DEGRADATION Chromatography can be used to identify the PTH residue generated by each cycle. If the released amino acids are identical with respect to molecular weight (for example, isoleucine and leucine have a molecular mass of 113 Da), they can be identified by different retention time. One cycle of the Edman degradation (cleavage of an amino acid from a peptide + identification) is carried out in less than 1 hour. By repeated degradations, the amino acid sequence of about 50 residues in a protein can be identified. PROTEIN SEQUENCING STRATEGY Peptides longer than about 50-70 amino acids long cannot be sequenced reliably by the Edman degradation. Long protein chains need to be broken up into small fragments that can then be sequenced individually. Digestion is done either by endopeptidases such as trypsin or pepsin or by chemical reagents such as cyanogen bromide. Different enzymes give different cleavage patterns, and the overlap between fragments can be used to construct an overall sequence. Beckman-Coulter Porton LF3000G Automated Each cycle releases and derivatises one amino acid from the protein or peptide's N- terminus The released amino- acid derivative is then identified by HPLC The sequencing process is done repetitively PROTEIN SEQUENCER  Mass spectrometry Mass spectrometry can, in principle, sequence any size of protein, but the problem becomes computationally more difficult as the size increases. The protein is digested by an endoprotease, and the resulting solution is passed through a high pressure liquid chromatography column. At the end of this column, the solution is sprayed out of a narrow nozzle charged to a high positive potential into the mass spectrometer. The charge on the droplets causes them to fragment until only single ions remain. The peptides are fragmented and the mass-charge ratios of the fragments measured. Extremely accurate, but expensive Protein sequencing: relying on the protein data base The mass spectrum is analysed by computer and often compared against a database of previously sequenced proteins in order to determine the sequences of the fragments. Mass spectrometry & Edman degradation MS provides high throughput automation with more precise and powerful protein analysis. However, N- terminal sequencing by Edman degradation still continues to complement MS in difficult protein identifications. THANK YOU! Oligonucleotide/Peptide synthesis and microarrayer  Oligonucleotide Synthesis Peptide synthesis Learning Outcomes Microarrays- Types and Applications Oligonucleotide Synthesis Nucleotide Structure  Synthesis of Dinucleotide by Michelson and Todd (1955) 3’-O-acetylthymidine was phosphorylated with a phosphorochloridate, and the phosphate group was protected with a benzyl group.  Khorana’s Synthesis Approach (Phosphodiester Method) 5’-O-tritylthymidine and 3’-O-acetylthymidine 5’-phosphate are reacted in the presence of toluene-4-sulfonyl chloride (TsCl) or N1,N3- dicyclohexylcarbodiimide (DCC) in a pyridine solution. The removal of the trityl and acetyl group yields the dinucleotide.   Proceeds in the 3′- to 5′-direction (opposite to the 5′- to 3′-direction of DNA biosynthesis in DNA replication) PHOSPHORAMIDITE  One nucleotide is added per synthesis METHOD cycle.  Solid phase oligonucleotide synthesis  (1) Detritylation PHOSPHO- (4) (2) RAMIDITE Oxidation Coupling METHOD (3) Capping  Step 1 - Detritylation The cycle is initiated by removal of the 5'-DMT (4,4'-dimethoxytrityl) protecting group of the solid-support-linked nucleoside (contains the terminal 3' base of the oligonucleotide). The 5'-DMT prevents polymerization of the nucleoside during functionalization of the solid support resin. The 5'-DMT protecting group is removed by TCA (trichloroacetic acid) in the solvent dichloromethane.The products include the 3' terminal nucleoside with a free 5'-OH and a DMT carbocation (an ion with a positively charged carbon atom). The nucleoside proceeds to step 2 in the synthesis while the DMT carbocation absorbs at 495 nm and thereby produces an orange color that can be used to monitor coupling efficiency.  Step 2 - Coupling Once the DMT has been removed, the free 5'-OH of the solid-support-linked nucleoside is able to react with the next nucleoside, which is added as a phosphoramidite monomer. The diisopropylamino group of the incoming phosphoramidite monomer in the solvent acetonitrile is ‘activated’ (protonated) by the acidic catalyst ETT [5-(ethylthio)-1H-tetrazole]. The products include a dinucleoside with a phosphite triester linkage and a free diisopropylamino group.  Step 3 - Capping Since 100% coupling efficiency is impossible, there are always some solid- support-linked nucleosides with unreacted 5'-OH. If not blocked, these hydroxyl groups will react during the next cycle, and hence, lead to a missing base. The accumulation of these deletion mutations through successive cycles would create a complex mixture of ‘shortmers’ that are difficult to purify. Capping is required to prevent shortmer accumulation. Acetic anhydride and N- methylimidazole react to form an intermediate in the solvent tetrahydrofuran, which contains a small quantity of pyridine. The product is the solid-support- linked nucleoside with an acetylated 5'-OH (pyridine maintains a basic pH thereby preventing detritylation of the phosphoramidite monomer by the free acetate / acetic acid).  Step 4 - Oxidation The phosphite triester formed during the coupling reaction is unnatural and unstable; therefore, it must be converted to a more stable phosphorus species prior to the start of the next cycle. Oxidation converts the phosphite triester to the stable phosphate triester. Oxidation of the phosphite triester is achieved with iodine in the presence of water and pyridine. The product is the phosphate triester, which is essentially a standard DNA backbone with a β-cyanoethyl protecting group on the free oxygen.  End of Synthesis - Cleavage Cleavage is necessary so that the free 3'-OH may take part in biochemical reactions, such as extension by DNA Polymerase during PCR when the oligonucleotide serves as a primer. Ester hydrolysis of the linker (and, simultaneous removal of the solid support) is carried out by treatment with concentrated aqueous ammonia. The product is the oligonucleotide with a terminal, free 3'-OH.  End of Synthesis - Deprotection After cleavage, the solution of oligonucleotide in concentrated aqueous ammonia is heated to remove protecting groups from the bases and phosphates.  Deprotection - Bases While thymine does not require a protecting group, adenine, cytosine, and guanine do since they contain exocyclic primary amino groups. The protecting groups must be removed so that proper hydrogen bonds between the oligonucleotide and the target nucleic acid may form. The oligonucleotide in concentrated aqueous ammonia is heated. The protecting groups include: N(6)- benzoyl A, N(4)-benzoyl C, and N(2)-isobutyryl G. The product of the reaction is fully-deprotected A, C, and G bases.  Deprotection - Bases In addition to the standard protecting groups, the labile dimethylformamidyl G and the ‘ultramild’ protecting groups can be used for modified oligonucleotides that are sensitive to ammonia. The dimethylformamidyl protecting group is typically removed in concentrated aqueous ammonia via heating but in significantly less time than is the isbutyryl group. The ultramild protecting groups include: N(6)-phenoxyacetyl A, N(2)-acetyl C, and N(2)- isopropylphenoxyacetyl G. They are typically removed at room temperature in a concentrated aqueous ammonia / methylamine solution.  Deprotection - Phosphodiester Formation The β-cyanoethyl group on the free oxygen of the phosphate must be removed to convert it from a phosphate triester to a phosphate diester (phosphodiester). The cyanoethyl groups are removed in concentrated aqueous ammonia via β- elimination (atoms or groups are lost from adjacent atoms). The reaction is quick because the hydrogen atoms on the carbon adjacent to the electron- withdrawing cyano group are highly acidic. The products are the oligonucleotide with a native phosphodiester backbone and acrylonitrile.  H-Phosphonate Method Following acid-mediated removal of the 5'-DMT protecting group, condensation is carried out by addition of an appropriately protected 2'-deoxy or ribonucleoside H- phosphonate monoester (20) and an activating agent (pivaloyl chloride/adamantoyl chloride). The product of this reaction is the H-phosphonate diester (21). Unlike the phosphite triester, this linkage is stable to the acidic conditions (3% trichloroacetic acid in dichloromethane) required for removal of the 5'-DMT group. Thus it is unnecessary to carry out oxidation at every cycle. Chain elongation therefore consists of two steps. Oxidation to the phosphodiester with aqueous iodine is completed following oligonucleotide synthesis. After the oxidation step, aqueous ammonia is used to remove nucleobase amide protecting groups and to cleave the oligonucleotide from the support.   Antisense oligonucleotides  Small interfering RNA  Primers for DNA sequencing and amplification  Probes for detecting complementary Applications DNA or RNA via molecular hybridization  Tools for the targeted introduction of mutations and restriction sites  Synthesis of artificial genes  Oligo therapeutics/gene therapy  DNA and RNA vaccine   https://www.sigmaaldrich.com/MY/en/technical- documents/technical-article/genomics/pcr/dna- oligonucleotide-synthesis  https://www.atdbio.com/content/17/Solid-phase- oligonucleotide-synthesis  https://www.biolytic.com/t-introduction-to-oligo- References oligonucleotide-dna-synthesis.aspx  https://www.mt.com/int/ar/home/applications/L1_A (Oligonucleotide utoChem_Applications/L2_ReactionAnalysis/oligonu cleotide-synthesis.html Synthesis)  https://www.biosyn.com/tew/the-chemical- synthesis-of-oligonucleotides.aspx  https://www.mdpi.com/1420-3049/18/11/14268/htm Peptide synthesis  Chemical synthesis of peptide: 1. Solution phase synthesis Liquid-phase peptide synthesis is a classical approach to peptide synthesis. It is useful in large-scale production of peptides for industrial purposes. 2. Solid-Phase Peptide Synthesis Strategy for  The Merrifield Method by Robert Bruce Merrifield Peptide Synthesis  Involves attaching one end of the peptide to a solid polymer.  Much quicker than classical synthesis, and leads to dramatically improved yields  Small solid beads, insoluble yet porous, are treated with functional units ('linkers') on which peptide chains can be built. The peptide will remain covalently attached to the bead until cleaved from it by a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid.   Step 1 - Attaching an amino acid to the polymer  The amino acid is reacted with a molecule known as a "linkage agent" that Solid-Phase Peptide Synthesis enables it to attach to a solid polymer, and the other end of the linkage agent is reacted with the polymer support. Common linkage agents are di- and tri-substituted benzenes.  Step 2 - Protection  An amino acid is an acid with a basic group at one end and an acid group at the other. To prevent an amino acid from reacting with itself, one of these groups is reacted with something else to make it unreactive.  Step 3 - Coupling  The protected amino acid is then reacted with the amino acid attached to the polymer to begin building the peptide chain.  Step 4 - Deprotection  The protection group is now removed from the acid at the end of the chain so it can react with the next acid to be added on. Steps 2 to 4 are repeated as each new amino acid is added onto the chain until the desired peptide has been formed.  Step 5 - Polymer removal  Once the desired peptide has been made the bond between the first amino acid and the linkage agent is broken with a 95% solution of trifluoro acetic acid (TFA)to give the free peptide.  Diagnosis and treatment  Epitope mapping Production of antibodies Applications Vaccine Drug delivery systems   https://www.ncbi.nlm.nih.gov/pmc/articles/PMC356 4544/  https://www.thermofisher.com/my/en/home/life- References science/protein-biology/protein-biology-learning- center/protein-biology-resource-library/pierce- (Peptide protein-methods/peptide-synthesis.html  https://pubs.acs.org/doi/10.1021/acs.joc.8b03001 Synthesis)  https://www.intechopen.com/chapters/66890 Microarrays   Microarrays are simply small glass or silicon slides upon the surface of which are arrayed thousands of features (usually What Are between 500 up to a million) Microarrays?  Multiplexed parallel- processing methods  High throughput  DNA microarrays Protein microarrays Tissue microarrays Cellular microarrays Types of Microarrays Chemical compound microarrays Antibody microarrays Carbohydrate microarrays   Microarrays are a technology in which 1000’s of nucleic acids are bound to a surface and are used to measure the relative concentration of nucleic acid sequences in a mixture via hybridization and subsequent detection of the hybridization events.  Specific DNA sequences are either deposited or synthesized in a 2-D (or sometimes 3-D) array on a surface in such a way that the DNA is covalently or non-covalently attached to the surface  A DNA array is used to probe a solution of a DNA Microarray mixture of labeled nucleic acids and the binding (by hybridization) of these “targets” to the “probes” on the array is used to measure the relative concentrations of the nucleic acid species in solution.  By generalizing to a very large number of spots of DNA, an array can be used to quantify an arbitrarily large number of different nucleic acid sequences in solution.  Basic types of microarrays (A) Spotted arrays on glass (B) Self assembled arrays (C) In-situ synthesized arrays A. With spotted arrays, a “pen” (or multiple pens) are dipped into solutions containing the DNA of interest and physically deposited on a glass microscope slide. Typically the glass slide surface is coated with something to help retain the DNA such as polylysine, a silane or a chemically reactive surface. B. Self assembled arrays can be created by applying a collection of beads containing a diverse set of oligos to a surface with pits the size of the beads. After the array is constructed a series of hybridizations determine which oligo is in what position on each unique array. C1 and C2. In-situ synthesized arrays can be produced by inkjet oligo synthesis methods (C1) or by photolithographic methods (light directed) such as used by Affymetrix (C2).   RNA is isolated from the sample of interest and enriched for messenger RNA. Applications  It is optionally amplified and labeled by any one of a number of methods and the resulting labeled sample is hybridized to a microarray. Gene Expression  The array is washed to remove unbound sample. Analysis  If the sample was labeled with biotin, the array is post stained with fluorescently labeled streptavidin and washed again.  The array is then scanned to measure fluorescence signal at each spot on the array.   A) Allele discrimination by hybridization – Oligos that are complimentary to each allele are placed on the array and labeled genomic DNA is hybridized to the array.  B) Two allele specific oligos are each tailed with a different universal primer (1 and 2) and hybridized Applications in solution to genomic DNA. A third oligo that is complementary to the same locus is tailed with a “barcode” sequence and a third universal primer (3). Polymerase is used to extend the allele specific primers across the genomic sequence and the Genotyping extended products are ligated to the third oligo. PCR is performed using primers complimentary to universal sequences 1, 2 and 3. The PCR primers complimentary to the universal sequences 1 and 2 are labeled with a unique fluorophore. The barcode sequence on the third oligo allows the PCR product to be uniquely detected on an array containing oligos complimentary to the barcode sequence. The use of multiple barcodes (one for each locus of interest) allows the assay to be multiplexed to sample many loci.   C) Arrayed primer extension (APEX) – In this assay, the array contains DNA oriented with the 5′ end Applications attached to the array and the 3′ end stopping one nucleotide short of the SNP. Genomic DNA is fragmented and hybridized to the array and the oligo on the array is extended in single nucleotide dye terminator sequencing reaction. Genotyping  D) This assay is similar to the APEX assay except that the oligo to be extended is on a bead and the single nucleotide that is added is labeled with a nucleotide specific hapten as opposed to a fluorophore. The haptens are then detected by staining with fluorescently labeled proteins that bind each hapten.   Determine what genes are active in a cell and at what levels  Compare the gene expression profiles of a control vs treated  Determine what genes have increased or decreased in during an experimental Usefulness condition  Determine which genes have biological significance in a system  Discovery of new genes, pathways, and cellular trafficking   Protein microarrays are prepared by immobilizing proteins onto a microscope slide using a standard contact spotter or noncontact microarrayer.  A variety of slide surfaces can be used. Popular types include aldehyde-and epoxy-derivatized glass surfaces for random attachment through amines, nitrocellulos, or gel-coated slides and nickel-coated slides for affinity attachment of Protein His6-tagged proteins. The last type was reported Microarray to provide 10-fold better signals than those obtained with other random attachment methods.  After proteins are immobilized on the slides, they can be probed for a variety of functions/ activities.  Finally, the resulting signals are usually measured by detecting fluorescent or radio-isotope labels..  Applications  Can be used to monitor protein expression levels or for bio- Analytical Protein marker identification, clinical diagnosis, or Arrays environmental/food safety analysis. A multivalent antigen is first caught by a capture antibody immobilized on the surface and then detected by a detection antibody. The label is usually tagged on the detection antibody. Applications  (i) To probe for various types of protein activities, including protein-protein, protein-lipid, protein-DNA, protein-drug, and Functional protein-peptide interactions Protein  (ii) To identify enzyme Microarrays substrates  (iii) To profile immune responses Protein:protein interactions   https://slideplayer.com/slide/1677136/  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC401 1503/  https://www.onlinebiologynotes.com/dna- References microarray-principle-types-and-steps-involved-in- cdna-microarrays/ (Microarrays)  https://www.future- science.com/doi/10.2144/06404TE01 CENTRIFUGE 1  Learning Outcomes 1. Principles of centrifuge 2. Centrifuge parts 3. Types of rotor 4. Types of centrifuge 5. Precautionary steps while using a centrifuge  Centrifuge A device for separating particles from a solution according to their size, shape and density. size - a large stone will sink more rapidly in water than a small one density - a ball made of steel will sink more rapidly than one of plastic shape - a cube will sink more rapidly than a coil.  Principle of Centrifuge Particles in a liquid medium settle into bottom of a container very slowly. centrifugal force When this container is spun about an axis at a high rotational speed, a centrifugal force is generated onto the particles, increasing the settling rate of the material in a liquid medium. The centrifugal force acts in a direction away from the center of the rotation axis. Centrifugal Force Centrifugal force G = ω2r. ω2 = square of angular velocity of the rotor (ω=radians s-1) and the distance (r, in cm) between the axis and centrifuge tube. However, centrifugal force is normally not measured in G, but in Relative Centrifugal Force (RCF), multiple of the earth’s gravitational field (unit = ×g). Eg. A relative centrifugal force of 5000 xg means a centrifugal force that is 5000 times stronger than the gravitational force exerts on a particle in a centrifuge.  Relative Centrifugal Force (RCF) RCF = 1.118 x 10-5 r N2 (unit ×g) Where r = rotational radius (cm) N = revolutions per minute (rpm) Eg. if a centrifuge radius is 25 cm and the rpm is 1500 rev min-1, the RCF is about 664 xg. RCF and RPM Because centrifuges come in different designs with different rotors, two centrifuges that spin at same rpm might not generate the same magnitude of centrifugal force. RCF, on the other hand, is the universal and transferable unit for all centrifuges from various manufacturers. Nomogram  Centrifuge Parts Motor 1. Centrifuge part that generate the rotational movement by electricity. 2. The speed of a centrifuge is controlled by raising and lowering the voltage supplied to the motor. Central Shaft 1. Rotate driven by electric motor. 2. Hold rotor in place. Centrifuge Parts Buckets 1. Hold adapter and centrifuge tubes 2. Buckets hang vertically when the rotor is at rest and swing 90o to a horizontal position, under the influence of centrifugal force Adapters 1. Come in a variety of designs to hold centrifuge tubes of various volume size Rotor 1. Fixed to the central shaft. 2. Two major types of rotors are fixed-angle rotor (built with holes to hold centrifuge tubes) or swinging-bucket rotor (have buckets hang on it for holding centrifuge tubes).  Refrigerator System 1. To prevent denaturation of heat sensitive sample. 2. In high speed centrifuge, refrigerator system monitors only the rotor chamber temperature. 3. In ultracentrifuge, refrigerator system monitors the rotor temperature directly, ensuring more accurate and responsive temperature control. Vacuum System 1. to evacuate air from chamber to minimise any excessive rotor temperature being generated by frictional resistance between the air and the spinning rotor.  Breaking System 1. Is incorporated to provide rapid rotor deceleration. 2. Either an electrical breaking system that functions by reversing the polarity of the electrical current to the motor, or a mechanical brake. Imbalance Detector 1. Monitors the rotor during operation, causing automatic shutdown if rotor loads are severely out of balance Swinging-bucket Rotor Swinging-bucket rotor Fixed-angle rotor  Supernatant Pellet Swinging-bucket rotor Fixed-angle rotor  Swinging-bucket rotor Also called swing-out rotor. Centrifuge tubes will swing out to a horizontal position during centrifugation. Since centrifugal force is proportional to distance between the axis and centrifuge tube, a particle will experience a greater force the further away it is from the axis of rotation. Thus, a particle will tend to move faster as they sediment through a liquid medium. Particles have longer travel distance, give better separation, longer run time.  Fixed-angle rotor Centrifuge tubes are hold at an angle between 14-45o to the vertical during centrifugation. Particles also experience greater force as they sediment through a liquid medium. Particles have shorter distance to travel before colliding with, and pelleting on, the outer wall of the centrifuge tube. Shorter run time.  Centrifuge Classification by purpose of usage: a) Preparative Centrifuge b) Analytical Centrifuge Preparative centrifugation techniques are more commonly used in undergraduate course and in general laboratory. Preparative centrifugation is used for actual separation, isolation and purification of materials suspended in a liquid medium for subsequent biochemical investigation.  Analytical Centrifuge Used for studying and measuring the physical properties of the sedimenting particles, such as sedimentation coefficient, molecular shape and molecular weight. Samples are centrifuged in cells (centrifuge tubes with quartz window) that lie paralleled to the plane of rotation of rotor. Molecules/particles are observed by optical system during centrifugation. Sample Cell for Analytical Ultracentrifuge  Preparative Centrifuge Analytical Centrifuge Larger sample size can be used Uses small sample size (< 1 ml to 100 ml) (< 1 ml) No optical system Built in optical system to analyze progress of molecules during centrifugation Less pure sample can be used Uses relatively pure sample Generally used to separate whole Used to precisely determine cells, cell components, nucleic sedimentation coefficient and MW acids, bacteria and viruses for of molecules subsequent investigation Can be a low-speed, high-speed An ultracentrifuge with a built in or an ultracentrifuge optical system Preparative centrifuge may be classified into four major groups: 1. Microcentrifuge 2. Small bench centrifuge 3. High speed refrigerated centrifuge 4. Ultracentrifuge Microcentrifuges and microcentrifuge tubes Small bench centrifuges High speed refrigerated centrifuges  Ultracentrifuge Preparative and analytical Refrigerated chamber to prevent protein denaturation. Sophisticated temperature monitoring system that can continuously monitor rotor temperature. Tightly sealed, a vacuum pump to evacuate air from chamber to minimise any excessive rotor temperature being generated by frictional resistance between the air and the spinning rotor. Isolation of cellular organelles (Golgi membrane, microtubule filaments, microsomes, ribosomes), viruses, membrane proteins and so on. Ultracentrifuge  Precaution Steps While Using a Centrifuge Only suitably trained person may operate a centrifuge. Balance the load in a centrifuge carefully. Never overfill centrifuge tubes (don't exceed ¾ full). Never exceed the maximum speed for any rotor. Use only correctly fitted tubes – unfit tubes can rupture at too high a speed. Check compatibility of tube material to solvent (some solvents may cause the tubes to swell or crack in the rotor).  Precaution Steps While Using a Centrifuge Always remain by the centrifuge until it has reached the maximum speed and is running smoothly. Stop the centrifuge immediately if an unusual condition (noise or vibration) begins and check load balances Clean up spillages immediately, use 70% alcohol if necessary. Never attempt to open the lid of a centrifuge or slow the rotor by hand or open the lid while rotor is in motion.  Balance the Load on Rotor The weight of tubes must be equal on the direct opposite sides of the rotor for the centrifuge to run efficiently, a blank should be used when there is odds number of tubes. The higher the spin speed, the more accuracy that is required between the sample and the blank weight Balance the Load on Rotor  Preventive Maintenance Rotor, buckets and shields should be examined regularly for signs of mechanical stress (cracks, corrosion). Dry centrifuge after use to prevent corrosion. Servicing and lubrication only by trained persons, according to the manufacturer’s instructions. Where necessary, keep a centrifuge log book. A log book must be kept for ultracentrifuge rotors because the rotors should be changed after the manufacturers' recommended running hours or years of service, whichever comes first. THANK YOU Centrifuge 2 Tsen Min Tze Ext 1241  Content 1. Cell fractionation 2. Differential centrifugation 3. Gradient centrifugation 4. Analytical ultracentrifuge Cell Fractionation process of producing relatively pure fractions of cellular components while preserving their function. Two main steps: 1. Disruption and lysis of cell membrane (homogenization). 2. Fractionation of the homogenate to separate the different populations of organelles (by differential centrifugation). Homogenization: to release the organelles and other cellular constituents as a free suspension of intact individual components Homogenization Homogenization: to release the organelles and other cellular constituents as a free suspension of intact individual components. Perform in suitable buffer and low temperature to protect the cell components. The resulting thick soup is called call homogenate. Several methods: 1. Sonication 2. Mechanical disruption 3. Osmotic lysis 4. Enzymatic digestion  Sonication The application of high frequency sound wave (ultrasound) (typically 20–50 kHz) to the samples in order to disrupt cell membranes and release cellular contents. The sound waves are delivered using an apparatus with a vibrating probe that is immersed in the liquid cell suspension. Shock wave created by the sound wave will break cell membrane. Mechanical Disruption Membrane are disrupted by mechanical shearing force. Performed carefully to prevent the damage of organelles. Several methods: 1. Pestle homogenizer 2. Bead mill homogeniser 3. Force cells through small hole using high pressure (syringe and needle) Mechanical Disruption Precellys 24 Bead Mill Homogenizer Precellys 24 Bead Mill Homogenizer Force cells through a Pestle homogenizer Bead mill homogenizer small hole using high pressure  Osmotic Lysis Mix cells with a buffer with a low osmotic pressure. Suitable for some animal cells like blood cells. Water will tend to flow into the cells by osmosis, promoting their lysis and release of cell components. Usually use in conjunction with mechanical disruption methods. Enzymatic Digestion Enzymes provide a very gentle and specific means of disrupting cells to release their contents. Cell walls of bacteria – lysozyme Plant cell walls – cellulases Enzymes are generally commercially available. Large scale applications of enzymatic methods tend to be costly and irreproducible. Differential Centrifugation A common method to separate cells fractions by centrifugation based on size, shape and density. Cells are first homogenized and suspended in solution. Homogenate is then subjected to increasing centrifugal force cycles. Separation is based on size and density, with larger and denser particles pelleting at lower centrifugal forces. Produce crude fractions of subcellular components. Differential Centrifugation Differential Centrifugation 1. Nucleus fraction - Intact cells, nuclei, plasma membrane 2. Mitochondria fraction – Mitochondria, lysosomes and peroxisomes 3. Microsome fraction – microsomes (fragment of rough and smooth ER) and other small vesicles. 4. Ribosomes in cytosol – ribosomes, viruses, large macromolecules. Only crude fraction, not 100% pure  Centrifugation Preparative Analytical Gradient Differential Rate zonal Isopycnic Gradient Centrifugation Rate zonal gradient centrifugation (Velocity gradient centrifugation) Isopycnic centrifugation (Equilibrium density gradient centrifugation) Rate Zonal Gradient Centrifugation Prepare a shallow (sucrose) gradient solution. (Density of sample particles must be greater than that of the highest density portion of the sucrose gradient) Layer sample at the top of the gradient solution. Centrifuge just sufficient amount of time for the particle separation to occur. Rate Zonal Gradient Centrifugation Separation is dependent on the size and shape of particles, thus it is used to separate particles with similar density but different size (mass) and shape. Isopycnic Centrifugation Particles are mixed evenly with cesium chloride (CsCl2) solution. Centrifuge at a very high speed. The salt solution will form a density gradient. (density of the sample particles must fall within the limit of this density gradient.  Isopycnic Centrifugation Particles will move down the density gradient, just like in rate zonal centrifugation, depend on their S value. When a particle reaches a point where the density of the solution is equal to its own density, it stops moving further and form a distinct band. Particles are separated based on their density. Ficoll, glycerol, sodium formate, potassium acetate, cesium chloride, cesium acetate…. and many more. Isopycnic Centrifugation Example of Isopycnic Centrifugation Peripheral blood mononuclear cell (PBMC) isolation using Ficoll. To separate lymphocytes (+monocyte and platelet) from granulocytes and blood cells.  Rate Zonal Isopycnic Velocity gradient centrifugation. Density gradient centrifugation. Separation is based on s