QBM Final Exam Study Guide PDF

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Study guide that covers all QBM. Runs through the textbook, history of QBM and actual application of biological practices...

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Quantitative Biological Methods Final Exam Study Guide ! The final exam will focus on the major concepts and techniques learned in the course. It will consist of 100 questions, ~25% from each section (Chapters 1-6, 7-12, 13-18, 19-21). The chapter questions, homeworks, and practice exams are also great sources of material to study.

Overview of Biomedical Research Chapter 1 – Molecular Biology Overview • DNA and protein basics: Central dogma: DNA->RNA (transcription)->Protein(translation) • Molecular biology definition: Structure and function of macromolecules (proteins and nucleic acids) • Molecular Surface model: best representation of DNA/protein if you could see it • Four levels of a protein structure o Primary - amino acid sequence o Secondary - form alpha and beta sheets o Tertiary -three-dimensional with folded motifs o Quaternary - multiple peptide chains Chapter 2 – Laboratory Safety, Oversight, and Hazards ● OSHA (Occupational Safety and Health Administration): o Employers must inform employees of potential hazards. ● MSDS: Material Safety Data Sheet for chemicals ○ Chemical’s physical and chemical properties, toxic, carcinogenic, e.g. ○ Describes proper way of handling these chemicals ● Biosafety Levels: ○ BSL-1: Minimal hazards. ○ BSL-2: Organisms with potential hazards; proper protective equipment; waste disposed properly; most teaching labs and some research labs. QBM lab is BSL-2. ○ BSL-3: Pathogenic organisms that cause serious disease; proper ventilation; lab restriction; special protective equipment; some research labs ○ BSL-4: Dangerous pathogens that cause severe to fatal disease; no vaccines or treatments ● PPE: Personal protective equipment (Latex > Nitrile > Plastic > None) Chapter 3 – Micropipettes, Centrifuges, and Spectrophotometers ● Pipet: Accurate liquid transfer o Graduated Cylinder: 25ml-2L ○ Glass/Plastic Pipettes: 1ml-25ml, Mohr is for finer adjustments, serological ○ Transfer/Pasteur Pipette: For quick measurements, non-accurate ○ Micropipette: 0.1µl-1ml accuracy, the tip should be in 1 cm of liquid and held vertically ● Autoclave: device with high temperatures and pressure used to sterilize glassware and peptides ● Centrifugation: used to separate samples based on mass, shape and density. ○ Analytical: analytical purposes, perform sucrose/CsCl gradients, studies sedimentation; determines characteristics such as sample purity, molec weight o Preparative: Most common, separate materials; purify sample for further study. o Small bench top centrifuge: 1.5ml tube (Can balance based on volume) o Large Capacity: Use for large volumes (1 L), separation of bacteria from media o High Speed : Large volumes needing higher speeds

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Ultracentrifuges: For samples that require a lot of force such as purifying a virus for sample. Operated under vacuum. 80,000 rpm and 600,000 g. (Must be balanced within 0.1 grams) o Sucrose Gradient: Sucrose gradients contain layers of different w/v solutions which allow you to separate samples based on density. They are used in swinging bucket rotors. o Centrifuges can be refrigerated to prevent protein degradation Rotors: o Fixed Angle: Most common; Particles pellet to a side when centrifuged o Swinging Bucket: Does not create tight pellets; for sample analysis or running cesium chloride or sucrose density gradients. Used to separate by density. Clear (Polycarbonate) Bottles: Better visibility, less resistant to chemicals, can weaken/ crack Opaque (Polypropylene) Bottles: More resistant to chemicals and cracks Spectroscopy: Study of Radiation interacting with matter, can analyze molec structure. (can be electromagnetic or non-electromagnetic) Spectrometry: Measurement of the spectroscopy interactions allowing to determine molecular structures/concentrations Spectrophotometer: Measures the intensity of a specific wavelength as it passes through a sample and compares it to a blank o 5 Components: light source(tungsten or deuterium), monochromator, prism, sample compartment, detector Plate Readers: Quickly read µl volumes of hundreds of samples from a single plate (used in ELISAs)

Chapter 4 – DNA Quantification, Protein Quantification, and Enzyme Assays ● 260nm:Maximum DNA Absorbance ● 280nm:Maximum Protein Absorbance ● 340nm: CDNB assay (enzyme activity) ● 560nm: BCA assay (protein concentration) ● 595nm: Bradford Assay (protein concentration) ● 600nm: Bacterial growth (E. coli.) in LB media ● 750nm: Lowry/DC Protein Assay (protein concentration) ● Absorbance/Transmission and Concentration Equations ○ A = εcb (b is always 1 cm for our purposes) ○ A = log (1/T) or A= -log (T) ; T = 10-A ○ Concentration is proportional to absorbance and is inversely proportional to transmittance ● DNA Quantification: A260 of 1.0= DNA concentration 50 ug/ml ● DNA Purity: 260/280 Ratio > 1.5 ○ Protein Quantification: 280nm because of ringed amino acids tryptophan and tyrosine ○ Amino Acid Analysis: Most accurate way to determine protein concentration ○ Colorimetric Methods: Measure change in color ■ Bradford: Coomassie brilliant blue dye, Fastest, 595nm, high variability, can be used without a standard curve ■ Lowry/DC: Folin reagent, Cheap and Easy, Copper, 750nm ● DC Assay allows detergents to be used; it is a modified version of the Lowry Assay. Low variability, more accurate, takes longer than bradford ● BCA method: instead of folin reagent use BCA reagent and measure at 560 nm, fast, easy, stable, low protein to protein variability. ● Standard Curve: Correlate absorbance(Y-axis) to concentration (X-axis)

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○ Accounts for variations in absorbance between different concentration of samples Enzymes: Speed up reaction, lowers activation energy. Only affects rate. No effect on equilibrium. Active Site Conservation: Conservation across species shows the importance of an AA sequence, likely to be in an active site Enzyme Substrate Models: ○ Lock and Key: Enzyme and substrate fit perfectly ○ Induced Fit: Binding of substrates induces conformational changes Enzyme Kinetics: How much substrate is being converted to product in a given time by an enzyme ○ Rate: Velocity depends on concentration of substrates, temperature, pH, and cofactors. ○ Competitive Inhibitors: Bind to same site on enzyme as substrates bind (active site) ■ To overcome competitive inhibition, add more substrate ■ Can reach Vmax ○ Noncompetitive inhibitors: Bind to second site on enzyme causing conformational changes preventing enzyme-substrate interactions ■ Cannot be overcome; drugs designed after this ■ Can’t reach Vmax Enzyme Assay: Determine enzyme’s rate. Ex: CDNB Assay 340 nm Kinase Assay: Study enzyme that hydrolyses ATP to ADP (detect phosphorylation)

Chapter 5 – Measurements, Concentrations, Dilutions, and Buffers ● Daltons: 1 Dalton = 1 g/mol; 9 AA = 1 kDa and 1 AA = 112 Da ● Molarity (M): Amount of solute in moles/Liter, mM- millimoles/Liter, µM- µmoles/Liter ● Percent composition (v/v and w/v): ○ v/v: Solute and solvent are expressed in same volume units. Ex: 30% v/v ethanol is 30ml ethanol + 70ml of water. ○ w/v: Amount of solute expressed in grams. The amount of solvent expressed in mL. 30% w/v NaOH would be 3 grams NaOH in a 10 ml volume of water ● Dilution Equation: (M1V1 = M2V2) Units don’t matter as long as they match across the equal sign ○ Buffers: be able to prepare simpler buffers ○ Molarity desired (mol/L) * Volume desired (L)*Molecular Weight (g/mol)= Solute needed (g) ○ Process ■ 1. Add 80% of L (water) ■ 2. Add solid(s) and dissolve ■ 3. Add HCl (or acetic acid) dropwise to get correct pH (omit this step if only salt is added); if you pass the pH, throw the buffer away (do not add NaOH) ■ 4. Fill up to final volume with water ○ Exceptions ■ Stock solutions of Tris with a given pH can skip the third step if the pH of the stock matches the pH of the needed buffer. Chapter 6 – Laboratory Data, Experimental Design, and Research Ethics ● Protocol writing ○ Include everything you need to reproduce the experiment exactly ● Seminars, conferences, poster sessions ○ Seminars: Also known as journal clubs are usually held at the program or department level. Grad students and postdocs present another's work to practice presenting.

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Invited Seminar Series: Allows for a large group of scientists to see current research from an internal or external researcher, who may be given an honorarium (payment). ○ Conferences: Also known as symposiums, gatherings of experts in the field, which features posters and invited lecturers. Allows scientists to present their research to colleagues ○ Poster Sessions: Possible way for grad students to present their data. Can be on campus ○ Grants: Applied (to the NIH or private donations) for and what funds the labs. ○ Proposals: Must show 3 things- If work is relevant, current published work supports their line of thought, and that preliminary data supports their hypothesis. ○ NIH Review Process: Each submitted NIH grant is reviewed by a review board, the most sought after grant is the R01, which provides millions of dollars and funding for 3-5 years. Authorship, Publication, Citation Basics ○ Authorship: Order of authorship is determined by the principle investigator (PI) of the laboratory who earned the grant that supported the work. First author usually performed most of the work including writing the paper, last author is usually the PI ○ Publication: Begins with relevant title, authors, abstract, methods section, results, discussion, and references. Impact factor means how many times the article is cited in the following 2 years by other research publications. ○ Citation Basics: Cite everything that is not original to this paper including your own stuff from previous papers. Peer review process ○ Article is analyzed and criticized by scientists in the field prior to publication. Most use 3 anonymous peer reviewers; final decision is up to the journal editor. Experimental Design ○ Scientific method: ask a question, identify the subject, evaluate current knowledge, formulate a hypothesis, identify variables, come to a conclusion. Variables, controls (positive and negative) ○ Independent variable: Manipulated (time, initial amounts) ○ Dependent variable: Changes in response to the independent variable ○ Controls: Remain constant and act as a baseline ○ Positive Control: Used as the “normal” test and should produce expected, measurable results; ensures the experiment was performed correctly ○ Negative Control: Nothing should be observed, indicates if the reagents are contaminated. Experimenter bias, prevention, scientific rigor, double blind experiment ○ Experimenter bias: includes one subconsciously biasing their data. ○ Anecdotes: Bias based on personal experiences ○ Preventing bias can be done by using good controls, statistically reporting all data, undergo peer review process, holding scientific rigor, and double blind experiments. ○ Scientific Rigor: Ensures data and conclusions are supported across all tests ○ Double-blind experiment: The subjects as well as the doctors/scientists administering the drug/test do not know which is the control group and which is the experimental group. ○ Triple -blind: experimenter does not know which is control and experimental

Protein Purification, Quantification, and Analysis Chapter 7 - Protein Expression, Purification, Dialysis, and Storage ● Protein production overview ○ In vitro (cell free expression system): Only requires a ribosome with the mRNA of interest. Fast, simple, produces pure protein, BUT expensive and small amount of protein produced ○ Prokaryotic: Easiest to work with since they take up the DNA plasmid easily and produce ost protein, cheap, well established (E. coli) Inclusion bodies may formmisfolded protein aggregates that are not biologically active - harsh chemicals (chaotropic salts) required to denature, refold, and resolubilize with low success rate ○ Yeast and plant cells: Eukaryotic cells that produce large amount of properly folded protein ○ Baculovirus: Infect cells with gene of interest. Efficient, large expression of proteins (5000 amino acids) but expensive and takes times ○ Mammalian cells: Ensures that the product is in its native state. Good for human protein. ● E. coli cell lines ○ BL21 cells - carry the T7 RNA polymerase gene ○ B834 cells - methionine auxotrophs ● Protein production and types of production systems available ○ Lac Operon: lactose absent = lac operon is “off” ● Repressor binds to the promoter ● Prevents the polymerase from transcribing the mRNA ● lactose present = lac operon is “on” polymerase can bind ○ Ptac expression vector (pGEX) ■ Simple system that has a Ptac promoter that is silenced by the lac repressor ■ Turned on by the addition of IPTG (lactose derivative) ■ Can be leaky and have low levels of protein expression ○ Phage expression vector (pET) ■ Addition of IPTG -> lac repressor is removed -> E. coli RNA polymerase and ribosomes transcribe/translate T7 RNA polymerase -> T7 RNA polymerase targets the T7 promoter on the plasmid -> transcribing the gene of interest -> E.coli ribosomes = Protein ■ 2 step system -has tighter control and therefore not as “leaky”. ● Cell lysis techniques ○ Mortar and pestle (blender): physically breaking up eukaryotic cells or organs. ○ Freeze/thawing: 3 cycles since ice crystals will disrupt cell membranes. ○ Chemicals: detergents can be used to disrupt the bacterial cell wall or cause osmotic shock, but these can interfere with some downstream techniques such as the Bradford ○ Enzymes: can be used to disrupt cell membranes. Must be purified out after lysis. ○ Sonication: using sound waves to break up cell walls and membranes. ○ Tissue homogenizer: used to break down cells by shearing or spinning at a high rpm but limited to eukaryotic cells. ○ French press: (40,000 psi) places cells under high mechanical pressure to destroy the cell wall causing the internal proteins and DNA to be released into the buffer. BEST ● Protein Purification ○ Centrifugation: separate contents leaving a supernatant and a pellet.

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Ammonium sulfate cut: aka salting out is controlled precipitation where proteins are gently removed from buffer and stabilized. Used for discovery of proteins in native state. Requires large quantities of samples. ○ Column Chromatography: loading a sample onto a column that contains beads that help separate the proteins and are then identified by a chromatogram. Detergents and chaotropic salts ○ Detergents: used to resolubilize the sample when working with membrane proteins ○ Chaotropic salts: Guanidine and urea unfold the tertiary structure of the protein back to its linear state, used for inclusion bodies Dialysis, protein concentration, protein storage ○ Dialysis: proteins are placed in a special membranous tubing with small holes that only allow the buffer through. Used to purify large proteins and acts as a buffer exchange ○ Protein concentrating: Removing some buffer from sample while keeping the protein ○ Lyophilization: Samples are dried under vacuum to a fluffy powder; can be stored for years ■ Protein Storage: Stable for 1 month at 4°C due, May be frozen for a single time ■ EDTA to chelate metals and inhibit proteases, β-Me to prevent oxidation of cysteines, Sodium azide to inhibit bacterial growth, propelyne glycerol to prevent ice crystal growth (cryoprotectant)

Chapter 8 - Size Exclusion, Ion Exchange, and Affinity Chromatography ● SEC overview: ○ Separation occurs through porous beads with an exclusion size. Proteins larger than the exclusion size fall through the column the fastest in the void volume (flow through). Then medium size proteins flow out and finally the smallest proteins and salts do To increase column resolution one can use longer columns and smaller beads. ● Relationship between SEC, the UV chromatogram, and SDS-PAGE ○ Chromatogram: Chart with the plotted absorbance for each protein fraction at 280 nm ○ SDS-PAGE is done after the SEC to verify the presence of the wanted protein and its purity ● IEX overview ○ Separates proteins based on their electric charge. Proteins with same charge as the beads or neutral flow through first and then the charges opposite to the beads flow through last ○ Proteins bound to column are eluted with NaCl which is measured in millisiemens. ● Relationship between pH, pI, and protein charge ○ The pI (determined by primary structure) is the pH at which there is an overall net neutral charge on the molecule. ○ If the pH is above the pI, there is a net negative charge on the molecule. (Anion exchange) ○ If the pH is below the pI there is a net positive charge on the molecule. (Cation exchange) ● Anion vs. cation exchange (if pI is 9, and pH is 7, what type of column do you use? etc.) ○ Anion exchange chromatography: exchanging negatively-charged anions using beads with a positive charge. The buffer has to have a higher pH than the pI of the protein (pH>pI) ○ Cation exchange chromatography: exchanging positively-charged cations using negative charged beads. Buffer has pH lower than the pI of the protein (pHCentrifuge the lysate to separate cellular fractions->Add proteinase K to degrade proteins(including nucleases) ->Use salts/organic solvents ○ EDTA: Chelates metals and inhibits DNases; not RNases ○ Phenol-chloroform extraction: Organic solvent to extract DNA from impurities (phenol-aqueous layer on top-DNA/RNA, chloroform-organic layer on bottomproteins/debris) ○ Alkaline lysis: SDS breaks phospholipid bilayer and NaOH dissolves structural proteins ○ Silica-based methods: Absorption of DNA to beads in presence of chaotropic salts ○ Difference between DNA and protein purification: ■ DNA doesn’t need stacking gel and has a built in charge-to-mass ratio, (no SDS) ■ DNA is not degraded like proteins, so it can be used for downstream applications ■ Small sample of DNA needed compared to protein ● Purpose of a miniprep: Lyse bacteria and remove everything (including genomic DNA) to collect very pure plasmid DNA ● DNA quantification, 260/280 ratio ○ Spectrophotometer, plate reader, or Nanodrop at 260 nm ○ “Pure” DNA sample: Absorbance 260/280 > 1.5 ● RNA purification overview, how does it differ from DNA purification? ○ RNA is less stable than DNA because of 2’ hydroxyl group that promotes degradation (RNases are not inhibited by EDTA) ○ RNase: Found everywhere, withstands autoclaving ○ β-ME: RNase-inhibitor ○ Chomczynski and Sacchi method: Guanidinium isothiocyanate denatures proteins and inactivates intracellular RNases, involves phenol-chloroform extraction, precipitation ○ polyT coated magnetic bead method: mRNA has polyA tail ● Agarose gel electrophoresis – large vs. small bands, circular vs. supercoiled ○ Thick band: High concentration; Thin band: Low concentration ○ Circular DNA: Runs slowest ○ Supercoiled DNA: Runs fastest ● Gel extraction, why can it be performed on DNA but not protein? ○ Can’t be performed with protein because proteins are boiled and denatured (will not refold properly) and protein techniques require more sample ○ Cut DNA bands out of agarose gel on a UV transilluminator ○ Purified from gel by miniprep column or freeze squeeze method ● DNA visualization – EtBr, Sybr Green, autoradiography advantages/disadvantages ○ EtBr (Ethidium bromide) ■ Intercalates DNA (Carcinogenic) ■ Can be incorporated into gel or the gel can be soaked in it ■ Easiest and most reliable (nanogram detection) ○ SYBR Green ■ Binds to DNA and is safer than EtBr ■ Lower intensity on visualization (microgram detection) ○

Autoradiography ■ Radioisotopes incorporated into DNA and detected with X-ray film

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Advantage: Most sensitive Disadvantages: Time consuming, radioactive molecules, special film developers

Chapter 14 - Recombinant DNA Technology ● Restriction enzymes, digestion patterns on a gel (linear vs. circular DNA) ○ Restriction Enzymes: aka restriction endonucleases, Many bind to DNA and digest it at specific DNA sequences REs are also used by bacteria as a defense against bacteriophages. ● Buffer tables, star activity ○ Buffer tables (Restriction Enzyme Table): useful when determining if two enzymes have ...