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BSC 3403
Exam 1
| Term | Definition |
|---|---|
| Molecular biology | the study of DNA and proteins and their interactions, and the molecular information transfer within a cell. |
| Recombinant DNA technology | the process of taking a gene from one source (such as human DNA) and joining it with another piece of DNA (such as a bacterial plasmid) and inserting that DNA into bacteria for further study and manipulation. |
| DNA (deoxyribonucleic acid) | contains two strands of nucleic acids antiparallel to one another; AT; GC; negatively charged phosphate backbone 5'-3'; nucleotide DNA bases are held together by hydrogen bonds and base stacking. |
| Central dogma of molecular biology | DNA is copied by DNA polymerase during the S phase of cell division, and mRNA is transcribed from DNA by RNA polymerase. The ribosome reads this RNA message and translates it into protein. |
| Proteins | consist of amino acids linked by a peptide bond between the carboxyl terminus of one amino acid and the amino terminus of the next; synthesis by the ribosome is from the N- to C-terminus; 22 amino acids used by most organisms to make all of its proteins. |
| Primary structure | a protein’s amino acid sequence; |
| Secondary structure | alpha helices and beta sheets are the most common; |
| Tertiary structure | further folding into a complex, 3D structure; |
| Quaternary structure | multiple polypeptide chains interacting with one another; best represented by the molecular surface (solvent-accessible surface representation). |
| probes | can be used to determine the presence or absence of an allele, which can indicate a predisposition to a specific disease; are useful in generating pedigrees, paternity testing, and forensics. |
| amino acids can be characterized by their properties | Acidic (negatively charged); Basic (positively charged); Hydrophobic; Nucleophilic; Small; Bulky; Aromatic; Many of these amino acids are further modified after synthesis with phosphates, sugars, and other groups for specific purposes. |
| Material Safety Data Sheet (MSDS) | states a chemical’s physical and chemical properties and any associated physical hazards, health hazards, the primary route of entry, exposure limits, and whether or not it is toxic, carcinogenic, flammable, or radioactive; |
| BSL-1 laboratories | have minimal potential hazards to laboratory personnel. |
| BSL-2 laboratories | work with organisms that are of moderate potential hazard and researchers must be trained with proper safe handling, protective equipment must be worn, and waste must be disposed of properly. |
| BSL-3 laboratories | work with pathogenic organisms that can cause serious disease. Labs must be properly ventilated, access is restricted, and special protective equipment must be utilized. |
| BSL-4 laboratories | work with dangerous pathogens that cause severe to fatal disease for which vaccines or treatments are not available. |
| Bacteria and other organisms must be | killed with 10% bleach or autoclaved before discarding |
| Needles | should not be recapped before discarding as this can lead to accidental sticking. |
| Research laboratories must | follow the rules set forth by the Occupational Safety and Health Administration (OSHA), which states that employers must inform employees of potential hazards, and that every hazardous substance has a MSDS |
| All glassware must | be cleaned with soap, a scrub brush, and multiple rinses with warm water followed by a final rise in deionized water (ddH2O) to remove any residual ions |
| Acetone | is not used to rinse glassware in molecular biology laboratories because it can interfere with reagents and damage DNA and proteins |
| Autoclave | a device which uses high temperature and pressure to fully sterilize glassware, pipet tips, and cell media. |
| Centrifuge | a device that spins samples held in tubes in a rotor at very high speeds and at many times the force of gravity (g). |
| Preparative centrifuges | used to prepare samples for further use; separate molecules and can help to isolate or purify a sample; small bench top (13,000 rpm, 7000g); Large-capacity refrigerated (6000 rpm, 6500g); High-speed refrigerated(25,000rpm, 60,000g); balance within .25g |
| Analytical centrifuges | here the sedimentation characteristics of pure biological macromolecules and molecular structures can be analyzed; ultracentrifuges (80,000 rpm, 600,000g); operate under vacuum and laser to watch balance; balance within 0.1g |
| Fixed-angle rotors | allow for excellent pelleting cells from media or precipitated DNA from a solution. |
| Swinging-bucket rotors | do not create pellets (any sedimented material will be spread across the bottom of the tube) and are best for sample analysis or running cesium chloride or sucrose density gradients in an ultracentrifuge. |
| Clear bottles | allow for better visibility, but are less resistant to chemicals and can weaken and crack over time. |
| Opaque bottles | are used by researchers most often because of the disadvantages of clear bottles. |
| Electromagnetic radiation | a type of energy (a self-propagating wave) that is transmitted through space at enormous velocities and includes visible light, heat gamma-rays, X-rays, UV light, infrared light, microwaves, and radio waves. |
| Spectroscopy | the study of this [electromagnetic] radiation interacting with matter, and can be used to analyze molecular structures or dynamics through absorbance, emission, and scattering. |
| Electromagnetic-based spectroscopy | includes those using visible, ultraviolet, and infrared light. |
| Spectrometry | the measurement of these interactions and allows one to determine molecular structure or the concentrations of solutions and can be measured wither qualitatively or quantitatively. |
| Absorbance | a characteristic of a substance that can be used to quantitatively determine the amount of substance in a solution. |
| Spectrophotometry | involves measuring a certain wavelength using prisms or gratings and measures the spectral properties of molecules. |
| Colorimetry | the conversion of a molecule to a colored compound by a chromogenic (color-forming) reaction; preferred over spectro. because this allows the measurement of the sample at other wavelengths that won’t be affected by DNA absorbance if the sample is impure |
| Spectrophotometers | devices used to measure the intensity of a specific wavelength of light as it passes through a sample. |
| conservative DNA replication | the original two strands remain together |
| semi-conservative DNA replication | the strands unwind and are used to make hybrid daughters of both old and new DNA |
| dispersive DNA replication | DNA is randomly distributed in the daughter strands |
| 1 Svedberg= | 10^(-13) seconds, relates to sedimentation during centrifugation |
| 260 nm | DNA absorption/concentration (special cuvettes required) |
| 280 nm | Protein absorption/concentration (special cuvettes required) |
| 340 nm | GSH-CDNB in the CDNB Assay, NADH (enzyme assays) |
| 560 nm | BCA Assay (protein concentration) |
| 595 nm | Bradford Assay (protein concentration) |
| 600 nm | Bacterial growth in LB media |
| 750 nm | Lowry/DC Protein Assay (protein concentration) |
| Absorbance (A)= | 2-log(%) = -logT= εcl |
| Transmittance (T)= | 10^(-A)=I/I_0 |
| The A260 of a sample is used to determine the concentration of DNA | according to a standard conversion factor in which the A260 of 1.0 = a DNA concentration of 50 μg/ml. |
| The most accurate technique for determining protein concentration is amino acid analysis | amino acid hydrolysis followed by chromatography but is unrealistic for routine protein concentration determination as it must be sent to a specially equipped laboratory and the costs are high (hundreds of dollars per sample) and a 2-5 day turnaround |
| Bradford Assay (Colorimetric protein quantification) | uses a dye called Coomassie Brilliant Blue that changes color from red (465 nm) to blue (595 nm) as the dye (VW) binds to proteins; nonlinear over wide range; 2x the variability than others; some compounds are incompatible in it; must be diluted; stnd crv |
| Biuret Method (Colorimetric protein quantification) | simplest method; involves copper (II) binding to the peptide nitrogen of proteins, which absorbs light at 550 nm; not very sensitive and does not work well in many common buffers, such as Tris |
| Lowry Method (Colorimetric protein quantification) | uses copper binding to peptide bonds; a folin reagent is added which becomes reduced by the Cu+ and turns blue, which is measurable at 750 nm; cheap and effective; low variability; can be detergent compatible; must be diluted; standard curve; |
| BCA Method (Colorimetric protein quantification) | similar to the Lowry Method but instead of the folin regent, BCA reagent is sued and measured at 560 nm; fast, the reagents are stable, and it has very low (~15%) variation between different proteins. |
| standard curve (Bradford, Lowry, and BCA Assays) | should always be used with a standard curve, which allows one to accurately correlate absorbance in the spectrophotometer to protein concentration; BSA can be used to create a standard curve; can be used to determine the [protein] of an unknown protien |
| Enzymes | are proteins or RNA that are essential to cell function by increasing the rate of a reaction; highly specific with respect to the substrates, single func. group can alter inter; very tightly regulated by cells; named based upon the reaction they catalyze; |
| Free energy (ΔG) | is a measure of the difference in energy between the substrates and products, and the change in free energy dictates whether a reaction will be energetically favorable. |
| A negative free energy represents | a thermodynamically favorable, spontaneous reaction (exothermic). |
| A positive free energy represents | a thermodynamically unfavorable reaction that requires an input of energy (endothermic). |
| Active site | is often a cleft or crevice on the surface of the enzyme which enhances the binding of the substrate, which interact with the active site via multiple weak forces; may require cofactors (metal) or coenzymes (organic molecules) to help reaction |
| the Lock and Key Model | proposed by Emil Fischer in 1894, the shape of the substrate and the active site of the enzyme fit together like a key into its lock (complementary); does not explain transition state stabilization. |
| Induced Fit Model | proposed by Daniel E. Koshland in 1958, the binding of substrate induces conformational changes in the enzyme, which bring the substrates together, and stabilizes the transition state. |
| Enzyme kinetics | the study of an enzyme’s rate, or how much substrate is converted to product in a given amount of time. |
| K_m= | ((K_(-1)+K_2)/K_1 ; A high Km indicates weak substrate binding; A low Km indicates strong substrate binding |
| v= | (V_max*[S])/(K_m+[S]) |
| Competitive inhibitors | bind to the same site on the enzyme that the substrate bind (the active site), this competing with substrates; affected by substrate concentration |
| Noncompetitive inhibitors | bind to a second site on the enzyme, causing conformational changes in the enzyme that prevent enzyme-substrate interactions; NOT affected by substrate concentration |
| enzyme assay | can be used to determine an enzyme’s rate. |
| A unit of restriction enzyme | is defined as the amount of enzyme required to cleave 1 μg of DNA in 1 hour in μl total volume |
| the purity of DNA can be determined | If the ratio of A260:A280 > 1.5, the sample is considered pure with few protein containments. |
| six major classes of enzymes | Oxidoreductases; Transferases; Hydrolases; Lyases; Isomerases; Ligases |
| To determine Km experimentally | a researcher can plot the effect of substrate concentration vs. enzyme rate; This data can be easier to analyze by using a Lineweaver-Burk Plot, where the x-intercept is -1/Km and the Y-intercept is 1/Vmax |
| kinase assay | Mix a protein kinase (or a lysate) with a substrate that can be phosphorylated and Radiolabeled (32P) ATP; Run sample on gel, develop, a band indicates radioactivity and therefore phosphorylation; Can be used to study signaling pathways or test inhibitors |
| Oxidoreductases | catalyze many oxidation-reduction reactions in which at least one substrate gains electrons, becoming reduced, and another loses electrons, becoming oxidized; includes dehydrogenases, oxidases, reductases and oxygenases |
| Transferases | catalyze the transfer of a specific functional group between molecules; includes phosphotransferases, acyltransferases that transfer fatty acyl groups, glycosyltransferases which transfer carbohydrate residues, and aminotransferases. |
| Hydrolases | cleave a covalent bond using water; includes proteases and nucleases |
| Lyases | catalyze the cleavage of bonds (C-C, C-O, C-S, C-N) by means other than hydrolysis or oxidation; includes aldolases, thiolases and decarboxylases. |
| Isomerases | catalyze the rearrangement of the bond structure within a single molecule. |
| Ligases | synthesize bonds between carbon atoms and carbon, nitrogen, oxygen or sulfur atoms in reactions that are usually coupled to the cleavage of the high energy phosphate of ATP or another nucleotide. |
| proteases | Proteases are the most widely used enzymes in the detergent industry. They remove protein stains such as grass, blood, egg and human sweat. |
| Lipases | remove fatty stains such as fats, butter, salad oil and sauces. |
| Amylases | residues of starch-based foods like potatoes, spaghetti, custards, gravies and chocolate. |
| zero order reaction | if the change in concentration of substrate produces no effect |
| 1st order reaction | if doubling the concentration of substrate causes the rate to double |
| 2nd order reaction | if doubling the concentration of substrate causes the rate to quadruple |
| Irreversible inhibition | inactivators that irreversibly associate with enzyme, activity of enzyme does not recover with dilution, usually covalent interaction |
| Reversible inhibition | inhibitors that can reversibly bind and dissociate from enzyme, activity of enzyme recovers when inhibitor diluted out, usually non-covalent interaction |
| dalton | the molecular weight of the protein; equal to an atomic mass unit, or 1/12th of the mass of a carbon-12 atom; approximately equal to the mass of a neutron or proton and therefore equal to 1g/mol |
| Solute | the molecule being dissolved, such as salt, protein, or DNA, and is usually measured in grams or moles. |
| Solvent | the solution responsible for dissolving the solute, and it is usually water and measured in liters. |
| Homogenous | when the solution is evenly distributed in the solvent. |
| Concentration | the amount of solute in any given amount of solution (the total volume of solute plus solvent) and is measured as molarity (moles/liter, or M). |
| To prepare solutions of known molarity | Amount needed (in grams)= M_w × desired concentration (M) × volume needed (L) |
| Solubility | the maximum amount of a substance that can be dissolved in the solvent. |
| Dilutions can be calculated using the following equation | (M1×V1)= (M2×V2) |
| Buffer solutions | usually consist of a mixture of a weak acid or base and its salt |
| R01 (a Research Project Grant) | can provide hundreds of thousands or millions of dollars to a laboratory and is funded for 3-5 years; New investigators (scientists earlier in their careers) are given special consideration. |
| The top scientific journals | are Nature, Science, the New England Journal of Medicine, and Cell, as determined by their impact factor, or total number of citations for each article in the following two years by other research publications. |
| 1st author in the authorship order | the graduate student or postdoc assigned to the project by the principle investigator |
| 2nd author in the authorship order | usually a close collaborator on the project |
| middle authors in the authorship order | laboratory technicians or other research scientists who contributed to the paper |
| penultimate author in the authorship order | often a supervising technical collaborator (such as another PI) |
| final author in the authorship order | the principle investigator of the laboratory and usually the corresponding author of the paper. |
| An independent variable | is that which is manipulated (time, initial amounts, etc.). |
| A dependent variable | changes in response to the independent variable and is observed and measured. |
| A control | acts as a baseline from which to measure changes in the dependent variable. |
| Positive controls | are used as the “normal” test and should produce expected, measurable results; These ensure that the experiment worked as designed. |
| Negative controls | should not be observed, and if they are seen it indicates that the sample or reagents are contaminated. |
| Experimenter bias | involves one subconsciously biasing their data (even computer software can be biased in its data selection or analysis). |
| To prevent bias | scientists use good controls, statistically analyze data, report all data (both good and bad) and any manipulations thereof, undergo the peer review process and experiment duplication, use double-blind experiments, and hold to scientific rigor. |
| scientific rigor | ensures the data and conclusions are consistent across all experiments, and helps to prevent experimenter bias. |
Created by:
JacobGant
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