7L Final Study Guide PDF

7L Final Study Guide Lab A - Scientific Method and the Memory Interference Test (MIT) Key Methods: 1. Hypothetico-Deductive Approach a. Def: series of steps that leads to robust conclusion about a particular problem i. Propos...

7L Final Study Guide Lab A - Scientific Method and the Memory Interference Test (MIT) Key Methods: 1. Hypothetico-Deductive Approach a. Def: series of steps that leads to robust conclusion about a particular problem i. Proposed by Karl Popper b. Starts with observations, forms hypotheses (𝐻1), and these tests them through experiments c. Uses statistical analysis to validate hypotheses 2. Falsificationist Procedure a. Def: a method of increasing the power of conclusions deduced using a hypothetico-deductive approach b. Introduces null hypothesis (𝐻0), predicting no effect or no difference c. Easier to disprove a hypothesis than to prove it 3. Good Hypothesis Characteristics: a. Specific: clearly defines groups and measures b. Testable: can be statistically rejected or retained after testing Definition of Inductive generalizations: Making generalizations based on one or more observations ○ For example, observing only white swans and concluding all swans are white Limitations ○ Are prone to revisions, as a single contradictory observation (e.g. seeing a black swan) can disprove the generalization ○ Tied to falsificationist procedure, emphasizing the difficulty in proving hypotheses and the ease of disproving them with a single counterexample Statistics: 1. Population: Entire group of interest (e.g. all humans in South America) 2. Sample: Subset of measurements from the populations 3. Descriptive Statistic  a. Def: describes the pattern of measurements and might be used to see whether samples are the same as expected 4. Parametric Data: a. Assumes normal distribution. b. Uses mean and standard deviation (SD) 5. Non-Parametric Data: a. Skewed or non-normal distribution. b. Uses median and median absolute deviation (MAD) 6. Measures of Central Tendency a. Mean: Average, used for bell-shaped distributions b. Median: Middle value, used for non-symmetrical data Not affected by extreme values (outliers) or skewed data making it a more reliable measure of central tendency than the mean c. Mode: Most frequent value d. Measures of Variability: Standard Deviation: Measures spread but for bell-shaped data Median Absolute Deviation (MAD): For all data types e. Parameter: show the central tendency f. How to Find the Interquartile Range (IQR) IQR = Q3 - Q1 g. Outliers: Use box plots to identify potential outliers based on the interquartile range (IQR): 1. Inner fence: Q1 - 1.5(IQR) or Q3 + 1.5(IQR) ○ Values outside the inner fence are suspected outliers 2. Outer fence: Q1 - 3(IQR) or Q3 + 3(IQR) ○ Values outside the outer fence are confirmed outliers.  7. Inferential Statistics a. Def: assess whether two samples are coming from the same population Compare two or more sample groups to determine if differences are significant b. Resampling (bootstrapping) is used in this lab instead of a t-test c. P-value: Probably of observing results as extreme or more extreme if 𝐻0 is true < 0.01: Statistically significant ; reject 𝐻0 ≥ 0.01: Not significant; fail to reject 𝐻0 Memory Interference Test (MIT) a. Def: computer program that uses either visual or auditory cues to test the subject’s memory b. Test Details: Five tests access recognition and reaction time Measures 1. Accuracy: Number of correct responses 2. Speed: Average reaction time Variability in results can arise from factors like sleep, stress, and time of day Lab B - Epidemiology & Lab Techniques Epidemiology: a. Def: study of diseases and populations at risk  Epidemic: higher-than-expected disease incidence in a population Pandemic: disease spread across large regions or continents b. Factors influencing disease spread: Environmental conditions Social and culture practices Genetic resistance (e.g. vaccination or natural immunity) Patient Zero a. Refers to the initial patient in an epidemiological investigation b. Term first applied to HIV spread; popularized despite flaws in early studies c. Identifying patient zero helps understand disease origin and spread Hidden Geometry in Contagion a. Mathematical models predicted disease spread based on: Infection and recovery rate Pathogen transport (e.g. via airports). b. Effective distance (flight connections) is a better predictor than geographic distance E.g. 2009 H1N1 influenza pandemic spread modeled effectively Assay a. Def: analyze samples for specific characteristics or a test used to qualitatively or quantitatively analyze a sample ○ Must include a standard/control and thorough consideration of the results obtained DNA assays: test for genes like CCR5-∆32 mutation (HIV resistance) Protein assays: measure protein levels (e.g. antibodies) 1. Detects immune response (e.g., post vaccination) 2. Uses 96-well plates for high-throughput analysis Requires control, precision, and thorough interpretation Protein gel electrophoresis a. SDS-PAGE : separates proteins based on their size and conformations ---> separating based on size can help identify which proteins are present b. Polyacrylamide gels are generally run vertically c. CCR5 mutant (~30kDa) vs. full-length (~46 kDa) Spectrophotometry  a. Def: used to accurately determine the concentration of DNA, RNA, or protein in a sample b. Uses light absorption at specific wavelengths c. The absorbance at any given wavelength is determined by measuring the difference in the signal with and without the presence of the compound of interest d. Serial Dilution: ○ Ensures accurate concentration measurements ○ Dilute solutions sequentially to detect absorbance ranges HIV: A Current Global Pandemic a. HIV Mechanism Virus infects T-cells via CCR5 protein Results in immune deficiency b. Natural Immunity CCR5-∆32 mutation prevents HIV entry into cells Potential therapeutic target for HIV treatment and prevention c. Assay Applications: Diagnose HIV Measure viral load Evaluate treatment effectiveness DNA Gel Electrophoresis a. Purpose: Separate DNA fragments by size using an agarose gel Detect CCR5-∆32 mutation or HIV integration Pipetting Techniques a. Pipettor basics: P20, p200, p1000 for specific volume ranges 1. The red indicates what pipette it is so for P20 a red number at the bottom and for a P1000 a red number at the top Avoid cross-contamination by changing tips after each use  P20: 2ul-20ul 1. Small yellow (or clear tips) a. 10’s , 1’s, 1/10/s respectively min max 0 2 2 0 0 0 P200: 20ul-200ul 1. Small yellow (or clear tips) a. 100’s, 10s, 1’s min max 0 2 2 0 0 0 P1000: 100ul-1000ul 1. Large blue (or clear tips) a. 1000’s, 100’s, 10’s min max 0 1 1 0 0 0  Experiment a. E. coli K-12 is a debilitated strain which does not normally colonize the human intestine Biological safety practices assume that these bacteria are potentially pathogenic despite along history of safe commercial use Considered a BSL Risk Group 1 agent and is not associated with diseases in healthy adult humans Lab C - Beta-galactosidase Assay β-galactosidase a. Hydrolyzes lactose into glucose and galactose b. E. coli only synthesizes this enzyme when lactose is present to conserve energy Lac Operon Regulation a. The lac operon is controlled by the lac repressor:  No lactose: Repressor binds to the operator, blocking transcription. Lactose present: Lactose binds to the repressor, causing it to fall off and allow transcription of lac genes (Z, Y, A) b. Lac operon: I gene Codes for the repressor Always active Repressor binds the operator which blocks the binding of RNA polymerase Lactose binds the repressor and frees the operator Its protein product, the lac repressor, is constantly produced Chromogenic Substrate a. ONPG (o-nitrophenyl-β-D-galactoside) substitutes for lactose b. Hydrolysis of ONPG produces o-nitrophenol, which is yellow (o-nitrophenol) and can be measured using a spectrophotometer Amount of o-nitrophenol produced is measured by determining the absorption Assay a. measuring how much of a given protein is produced per cell per unit time b. Indirectly measures enzyme activity by quantifying the product (o-nitrophenol) formed c. Optical density (OD) at 420 nm reflects o-nitrophenol concentration Miller Units Formula 𝑂𝐷420 Miller Units = 𝑇𝑖𝑚𝑒 𝑥 𝑉𝑜𝑙𝑢𝑚𝑒 𝑥 𝑂𝐷600 𝑂𝐷420: Measures o-nitrophenol production 𝑂𝐷600: Estimates cell density in the culture (cells/mL) Time: the amount of time that β-galactosidase and ONPG reacted together Volume: the volume of the sample removed from a culture Blank  a. provides a baseline optical density ---> contains all the ingredients except bacteria cells, glucose, and lactose (only contained Luria broth) The LB blank is used to measure the baseline optical density (𝑂𝐷600) of the sample Reagents 1. Luria broth a. provides essential nutrients for bacterial growth b. It is a nutrient-rich medium used to grow E. coli cultures 2. PopCulture Reagent a. A buffered mixture of detergents that perforates E. coli cell walls to release proteins into solution without denaturing them 3. Z Buffer a. Promotes the reaction between β-galactosidase and ONPG by optimizing the pH of the sample 4. 𝑁𝑎3𝐶𝑂2 (Sodium Carbonate) a. Stops the β-galactosidase reaction by increasing the pH from 7.0 to 11 b. Ensures that the yellow color (o-nitrophenol) remains stable for spectrophotometric measurement To calculate the absorbance of the undiluted stock solution Absorbance of stock solution=Absorbance of final dilution×Total dilution factor Lab D - Human Physiology Introduction a. Respiratory System: Breathing patterns and rates. b. Cardiovascular System: Heart activity and responses. c. Musculoskeletal System: Grip and muscle strength. d. Nervous System: Responses to various stimuli. Stations a. Wireless Heart Rate Monitor Measures heart rate changes during rest, exercise, and other conditions  Pace of heart contraction Measures the electrical activity transmitted by the heart muscle through the skin of your hands Fight and Flight Response ○ Triggered by the activation of the sympathetic nervous system in response to a perceived threat or stressor ○ This response prepares the body to either confront (fight) or escape (flight) from the threat ○ Effects: a. Increased heart rate and respiratory rate. b. Redistribution of blood flow to muscles. c. Release of glucose for energy. b. EKG/EMG Sensor Records electrical activity of the heart (EKG) and muscles (EMG) Detects an electrocardiogram (ECG or EKG) an electromyogram (EMG) 10 electrodes - 12 leads (directions) but in LS7L we only have 3 electrodes PQRS  P Wave: ○ Represents atrial depolarization, which is the electrical activity that triggers the contraction of the atria PR Interval: Measured from the beginning of the P wave to the beginning of the QRS complex ○ Represents the time it takes for the electrical impulse to travel from the atria to the ventricles QRS Complex: Represents ventricular depolarization , which is the electrical activity that triggers the contraction of the ventricles ST Segment: Represents the period between ventricular depolarization and repolarization ○ It is an isoelectric (flat) segment under normal conditions T Wave: Represents ventricular repolarization, or the recovery of the ventricles ○ It is typically a positive wave QT Interval: Measured from the beginning of the QRS complex to the end of the T wave ○ Represents the total time for ventricular depolarization and repolarization R-R Interval 60 Heart Rate (bpm)= 𝑅−𝑅 𝐼𝑛𝑡𝑒𝑟𝑣𝑎𝑙 (𝑠𝑒𝑐𝑜𝑛𝑑𝑠) c. Hand Dynamometer Measures grip strength and pinch strength Force of muscle contraction  d. Respiration Belt Tracks breathing patterns and rates using a force sensor Respiratory patterns Explain how you think carbon dioxide affects your respiration rate ○ Activation of chemoreceptors in the medulla oblongata e. Carbon Dioxide and Respiration (Student Question 2): Elevated CO₂ levels increase respiration rate via activation of chemoreceptors in the brainstem. Breathing Experiments: ○ Breath-holding: CO₂ buildup triggers the urge to breathe. ○ Rebreathing: Reintroduces CO₂ into the system, altering respiration rate. f. Tachycardia Is a high heart rate, defined as above 100 bpm at rest g. Bradycardia Is a low heart rate, defined as below 60 bpm at rest Lab E - DNA Isolation and Primer Design Introduction to mtDNA a. Mitochondrial DNA Small, circular DNA molecules located in mitochondria. Haploid: ○ Inherited only from the egg parent. Used to trace egg-parental lineage over generations.  High mutation rate due to lack of repair mechanisms. Contains a hypervariable region used for genetic studies. b. Haplogroups: Groups sharing similar mtDNA sequences with unique mutations Reflect ancient human migration patterns out of Africa Determined by key mutations compared to the Reconstructed Sapiens Reference Sequence (RSRS) c. Global Migration Patterns of Haplogroups Africa as the Origin: ○ Haplogroups L0, L1, L2, and L3 originate in Africa (~130,000–200,000 years ago). ○ Haplogroup L3 gives rise to M and N, marking migration out of Africa (~65,000–70,000 years ago). Migration to Asia and Oceania: ○ Haplogroup M moves eastward into Asia (~50,000 years ago). ○ Haplogroup Q reaches Oceania (~48,000 years ago). Migration to Europe: ○ Haplogroup N moves into Europe, diversifying into H, J, K, T, U, and others (~39,000–51,000 years ago). Migration to the Americas: ○ Haplogroups A, B, C, and D reach the Americas (~15,000–20,000 years ago), via the Bering land bridge. ○ Haplogroups B and A spread southward into South America (~3,000 years ago). Key Timelines: ○ Haplogroup dispersal aligns with archaeological and genetic data on human migration. ○ Mutations such as C16327T (haplogroup C) and G16129A (haplogroup Q) help trace movement. d. Phylogenetic Tree of Mitochondrial Haplogroups African Haplogroups (L0, L1, L2, L3): ○ Haplogroups L0 and L1 are the oldest and confined to Africa. ○ Haplogroups L2 and L3 are also African but show some migration patterns leading to global dispersal. ○ Mutation T16189C marks the evolution of L2. Out of Africa (M and N): ○ Haplogroups M and N branch from L3, marking the migration out of Africa. ○ Haplogroup M is dominant in Asia, Oceania, and the Americas. ○ Haplogroup N further diversifies into groups found in Europe, Asia, and the Americas. European Haplogroups (R, HV, J, T, U, K, W, X): ○ Haplogroup R gives rise to several European lineages. ○ Example mutations: a. T16189C: Associated with haplogroups HV, H, J. b. C16278T: Defines haplogroup T. c. T16311C: Defines haplogroups J and R. Asian and Oceanic Haplogroups: ○ Haplogroups B, F, Y, Q, and others are dominant. ○ T16217C: Found in haplogroup B. ○ A16227G: Marks haplogroup Q in Oceania. American Haplogroups (A, B, C, D): ○ Haplogroups A and B arise from N, while C and D arise from M. ○ Mutations like T16362C define haplogroup D  e. Regions in mtDNA Total size: 16,569 bp Focus on hypervariable segment I (HVSI) within the control region (600 bp), which accumulates mutations at a higher rate Concepts: Primer Design and PCR a. Primer Design: DNA has two strands: 1. Reference strand: Runs 5′ to 3′. 2. Complementary strand: Runs 3′ to 5′ (opposite direction). Forward Primer binds to the complementary strand. 1. Binds to the complementary strand (runs 3′ → 5′) and extends in the 5′ → 3′ direction Reverse Primer binds to the reference strand. 1. Binds to the reference strand (runs 5′ → 3′) and extends in the opposite direction. Must amplify the DNA sequence between key mutations. b. PCR Refresher: Steps: 1. Denature: Separate DNA strands (94°C). 2. Anneal: Bind primers to target sites (50–68°C). 3. Extend: Synthesize new DNA strands (72°C).  Produces billions of DNA copies through repeated cycles. To calculate the product size for a PCR reaction Product Size (bp)=Reverse Primer End Position−Forward Primer Start Position+1 c. Primers must be designed carefully to avoid problems with PCR. Primers should be 20 – 30 base pairs long (too short = not very specific, too long = difficult to bind to DNA). Melting temperatures between 55 – 80O C usually produce the best results Guanine and cytosine should make up 50 – 60% of the total base pairs (helps determine melting temperature). Primers should end (3’) in a G, C, CG or GC (G & C bind more tightly than A or T, so this will increase primer specificity). Long runs (four or more of the same base in a row) should be avoided (can cause the primer to bind in the wrong place). SNP Analysis and Haplogroups a. SNPs: (Single Nucleotide Polymorphism): Changes in nucleotide sequence at specific positions (e.g., C16069T). Used to define haplogroups and trace evolutionary history. Example: At position 16069 in the DNA sequence: 1. Reference (RSRS): Cytosine (C) 2. Mutated Version: Thymine (T) 3. This mutation is written as C16069T, meaning "C has changed to T at position 16069." Lab F - Agarose and Polyacrylamide Gel Electrophoresis SDS-PAGE (Polyacrylamide Gel Electrophoresis): a. Separates proteins based on size in their denatured state. b. Key features: SDS: 1. Detergent that denatures proteins and coats them with a uniform negative charge. 2. Ensures migration is proportional to size, not charge. Reducing agent: 1. Breaks disulfide bonds, further unfolding the proteins. c. Gel Matrix: Polyacrylamide forms a dense matrix ideal for small molecules like proteins.  d. Proteins migrate toward the positive electrode, with smaller proteins traveling farther. e. Limintation: Does not disrupt disulfide bonds unless combined with BME. Agarose Gel Electrophoresis: a. Used for separating DNA fragments. b. Confirm the presence, size, and purity of PCR-amplified DNA. c. Estimate DNA quality and concentration. b. Key features: ○ Agarose: Polysaccharide gel suitable for larger molecules like DNA. ○ GelGreen: Safer alternative to ethidium bromide, intercalates into DNA, fluoresces under blue or UV light. c. DNA migrates toward the positive electrode, with smaller fragments traveling farther. Calculate DNA Concentration: Formula: Concentration = 𝑂𝐷260​×50×dilution factor(100) Use spectrophotometry at 260 nm (DNA) and 280 nm (protein). 𝐴260 ○ Purity: Purity= 𝐴280 ​ (should be >1.8 for pure DNA) OR 𝑂𝐷260 ○ 𝑂𝐷280 Data Analysis a. Standard Curve for SDS-PAGE: Plot migration distance (cm) vs. molecular weight (kDa) of standards. Use the trendline equation to calculate unknown protein sizes. Fit data to an exponential equation to determine the size of unknown proteins: −0.5𝑥 1. MW = 123 x 𝑒 Comparison of Agarose and Polyacrylamide Gels Feature Agarose Gel (DNA) SDS-PAGE (Protein) Purpose DNA size verification Protein size estimation  Resolution Lower (good for large Higher (small size molecules) differences) Gel Structure Horizontal setup Vertical setup Safety Agarose is safe, GelGreen Acrylamide is toxic Safe before polymerization, SDS is harsh Visualization GelGreen fluoresces under AquaStain binds proteins LED/UV (blue stain) SDS: a. Sodium dodecyl sulfate, denatures proteins and adds a negative charge. GelGreen: a. DNA stain that fluoresces under blue/UV light. Polyacrylamide Gel: a. Dense gel for protein separation. Agarose Gel: a. Looser gel for DNA separation Staining: a. DNA: Visualized with GelGreen in running buffer. b. Protein: Stained with AquaStain to highlight bands. Spectrophotometry: a. Quantifies DNA concentration and purity using absorbance ratios. Why do smaller molecules migrate farther in electrophoresis? a. Smaller molecules can easily navigate through these pores, while larger molecules face more resistance. b. Smaller molecules experience less friction and resistance, allowing them to migrate faster and farther. What does a low A260/A280 ratio indicate about DNA purity? a. A260/A280 Ratio:  The absorbance at 260 nm (A260) measures nucleic acids (DNA/RNA). The absorbance at 280 nm (A280) measures proteins, specifically aromatic amino acids like tryptophan and tyrosine. b. Ideal Ratio: Pure DNA has an A260/A280 ratio of ~1.8. Pure RNA has an A260/A280 ratio of ~2.0. c. Low Ratio (