QBM Final Exam Review

CH1: Overview of Molecular Biology

Molecular Biology involves the study, analysis, and manipulation of DNA & proteins.
M.B. has its roots in early 1900s when geneticists and biochemists began to understand
genes & proteins.
Geneticists used bacteria and fruit flies to study genes; biochemists studied and
characterized the proteins those genes encoded.
1953: Watson & Crick determine the structure of DNA & proteins
= True beginning of M.B.
M.B. = 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 and
joining it with another piece of DNA and inserting that DNA into bacteria for further
study and manipulation.

DNA
2 strands Antiparallel 5->3 Directionality A/T & C/G
DNA copied by DNA Polymerase during S phase of cell division, and mRNA is
transcribed from DNA by RNA Polymerase. The ribosome reads this message and
translates it into protein. (CENTRAL DOGMA OF MOLECULAR BIOLOGY)
DNA probes can be used to determine the presence or absence of an allele, which can
indicate a predisposition to a specific disease
Probes useful for generating pedigrees, paternity testing, and forensics.
Small Scale: Genes can be knocked out to study their impact, or inserted via gene therapy
to cure a disease
Large Scale: Entire genomes can be sequenced and studied, and the sequence can be
compared between individuals or species.

Proteins
Consist of amino acids linked by a peptide bond between the carboxyl terminus of one
amino acid and the aminos terminus of the next.
Protein synthesis by the ribosome if from the N- to C-terminus.
Primary Structure: Amino Acid Sequence
Secondary Structure: alpha-helices and beta-sheets
Tertiary Structure: several motifs (alpha-helices, beta-sheets, etc) further folded into
complex
Quaternary: Interaction between multiple polypeptide chains.
Clinical Perspective- protein markers fors are important for disease classifications and
protein mutants often play a role in phenotype or disease states.
Most drugs interact with or inhibit protein function in some way, so knowing the
proteins function and impact on the cell is essential.
Proteins can be mass produced for medicinal use (insulin, vaccines, and certain
enzymes)
**p53 protein= tumor suppressor activated upon DNA damage or cell stress; binds to
DNA and activates transcription of the damage response genes.

CH2: Lab Safety, Oversight, and Hazards
Carcinogenic = cancer-causing
Occupational Safety and Health Administration (OSHA): Set rules for research
laboratories to follow. Also states that:
Employers must inform employees of potential hazards
Every hazardous substance has to have a Material Safety Data Sheet (MSDS)
MSDS: states a chemicals 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. Also lists
proper safe handling of the chemical and emergency and first aid procedures.

Centers for Disease Control (CDC) regulates researchers who work with microorganisms
that can cause disease by assigning each lab a Biosafety Level (BSL)
BSL-1: Minimal potential hazards to lab personnel
**BSL-2: these labs work with organisms that are of moderate potential hazard
BSL-3; these labs work with pathogenic organisms that can cause serious disease.
Must be properly ventilated, access is restricted, and special protective equipment
must be utilized.
BSL-4: these labs work with dangerous pathogens that cause severe to fatal
disease for which vaccines or treatments are not available.

Locally, each institution is regulated by internal oversight through a specific department
or Laboratory Safety Officer.
At UCF, the Department of Environmental Health and Safety provides a
comprehensive health and safety program and makes the campus a safe and
healthful place in which to live, learn, teach, work or visit.
DEHS also offers training classes and certifications required for specific
procedures (such as handling radioactive material) and chemical and biological
waste disposal. Also respond in event of emergency or to clean up waste spills.
Personal Protective Equipment (PPE) is essential in the lab for protection from hazardous
chemicals and pathogens.
Latex/Nitrile gloves (possibility of latex allergies)
Plastic gloves not acceptable
Special gloves for handling hot/cold, UV eye protection, specific sites for waste
disposal, and lab coats/pants sometimes required.
Decontaminate benches with EtOH
CH3: Micropipettes, Centrifuges, and Spectrophotometers
Most lab equipment that comes into contact with samples must be clean and sterile before
use.
Acetone NOT used to rinse glassware b/c it can interfere with reagents and
damage DNA and proteins.
Autoclave Device which uses high temperature and pressure required to fully
sterilize glassware and other equipment.
Transferring Liquids and Micropipetting
Volumes (~25ml to 2L) GRADUATED CYLINDER
Volumes (~1-25 to 25ml) GLASS/PLASTIC PIPETTES
Mohr pipette: Has pointed tip for finer measurements b/c drops will be smaller
Serological pipette
Transfer/Pasteur pipette: used for quick transfers when accuracy is not required.
Micropipettes (~0.1ul to 1ml) contains plunger, tip ejector, volume adjuster, volume
setting window, and disposable plastic tip.
Accuracy depends on calibration and proper technique (tip 1cm into liquid/
perfectly vertical
Centrifugation
Centrifuge: spins samples held in rotor at very high speed and at many times the force of
gravity (g)
Separate samples by mass, shape, and density.
Greatly increases rate of sedimentation.
Can be set by adjusting their g (also relative centrifugal force, rcf) or revs per minute
(rpm). Best to use g
Two types used in the lab:
Preparative: Used to prepare samples for further use. Separate molecules and can
help to isolate/purify a sample. Used to spin down tubes quickly, used in
minipreps and gel extractions of DNA, and for protein concentration. Usually
reach 13,000 rpm and ~7000g. Ex- Small bench-top centrifuge
Large-capacity refrigerated centrifuge: used with larger volumes (up to 1L) for
separation of large amounts of material like liters of bacteria from media. Usually
reach 6000rpm and ~6500g.
High-speed refrigerated centrifuge: used for large volumes needing even higher
speeds for the collection of microorganisms, cellular debris, organelles, or
proteins precipitated by ammonium sulfate. Sample volumes ares usually smaller
(~50ml tubes). Can reach about 25000 rpm and ~60000g. Samples must be
balanced within 0.25g of one another
Ultracentrifuge: useful for separating samples that require a lot of force. Operate
under vacuum to prevent frictional heat; also monitored by lasers to detect
unbalancing. Can reach 80,000 rpm and ~600,000g. Require samples to be
balanced within 0.1g of one another.
Analytical: can be used to determine a samples purity, molecular weight and size,
conformational changes, sedimentation and diffusion coefficients, stoichiometry,
equilibrium constant, thermodynamics, and absorbance or fluorescence. Also used
to perform sucrose or cesium chloride gradients to analyze or separate a sample.
Fixed-Angle Rotors: allow for pelleting of a sample
Swinging-Bucket Rotors: dont creat tight pellets. Best for sample analysis or
running cesium chloride or sucrose density gradients (5 % -> 25%) in an
ultracentrifuge.
Meselson-Stahl Experiment: Utilized swinging-bucket centrifugation to analyze DNA
replication.
Bacteria was grown in heavy (15-N) DNA, and switched to light (14-N) DNA,
then analyzed for various generations. DNA was originally denser, but as each
generation replicated the DNA became lighter. Demonstrated that DNA
replication is semiconservative. Figure on pg.22
Various types of bottles used.
Clear allow for better visibility, but are less resistant to chemicals and can weaken
and crack/break over time.
Spectroscopy & Spectrophotometry
Electromagnetic Radiation: a type of energy (self-propagating wave) that is transmitted
through space at enormous velocities, and includes visible light, heat gamma-rays, X-rays, UV
light, IR light, microwaves, and radio waves.
Spectroscopy: the study of this radiation interacting with matter; can be used to analyze
molecular structures or dynamics through absorption, emission, and scattering.
Spectrometry: the measurement of the interactions of spectroscopy. Can be measured
quantitatively or qualitatively.
Spectrophotometry: involves measuring a certain wavelength using prisms or gratings
and measures the spectral properties of a molecule.
Spectrophotometers: device used to measure the intensity of a specific wavelength of
light as it passes through a sample.
-These devices compare the amount of light transmitted through a sample to that
transmitted through a colored sample.
-Components: Light source (tungsten visible / deuterium UV);
Monochromator (selects particular wavelengths); prism or grating (disperses
light); sample compartment (holds cuvette in place); detector (uses phototubes
and photomultipliers to convert photons into electrical energy)
-Plate Reader: can quickly read microliter volumes of dozens or hundreds of
samples from a single plate
Colorimetry: converts a molecule to a colored compound by a chromogenic (color-
forming) reaction.

Chapter 4 DNA Quantification, Protein Quantification, and Enzyme Assays
-Only light of the correct energy for causing transitions from state to another is absorbed.
260nm: DNA
280nm: Protein
595nm: Bradford Assay / Coomassie Brilliant Blue when bound to proteins
600nm: Bacterial growth in LB Media
-Transmittance (T) = Incident light energy (I0) / transmitted light (I)
- Concentration = Absorbance = Transmittance
Transmittance decreases exponentially

Beers Law
A = 2 log(%T) = -logT = log (1/T) = ebc = ecl AND T = 10 ^ (-A)
A = absorbance or optical density (OD600)
e = Molar extinction coefficient (L * mol- * cm-)
-tryptophans X 5500 + tyrosines X 1490 + cysteines X 125
b / l = path length (usually 1cm)
c = concentration (mol/L)
DNA Quantification: @ 260nm (UV part of spectrum)
A260 = 1 [DNA] = 50 ug/ml
Purity: A260/A280 > 1.5
A230 = organic contaminants
A320 = sample turbidity or buffer absorbance

Protein quantification: @ 280nm (due to tryptophan and tyrosine)
-Proteins lacing tryptophan or tyrosine will have little or no absorbance at 280nm
Amino Acid Analysis: Most accurate method for determining protein concentration.
Protein is analyzed by amino acid hydrolysis followed by chromatography, where the proteins
identity and concentration are determined. Very expensive and takes various days, but very
accurate
Colorimetric methods
-Bradford Assay: fastest and simplest method. Uses coomassie brilliant blue
which changes color from red (465nm) to blue (595nm) as dye binds to proteins
(forms noncovalent/van der Waals complexes with proteins).
-Binding is relative to number of positive charges on the protein.
-Does NOT detect free amino acids or DNA; does NOT bind to proteins below
3000Da
-Requires STANDARD CURVE with each assay, HIGH variability
-Somewhat accurate and cost-effective
Lowry Method: uses copper binding to peptide bonds (little interaction with free
AAs). Uses a folin reagent which becomes reduced by Cu+ and turns blue
(measured at 750nm)
-Commonly used, inexpensive, easy, reproducible, LOW variability, more
sensitive than A280
-Must use STANDARD CURVE
-Sensitive to contaminants
DC Protein Assay: Modified Lowry Assay (detergent compatible)
-upper range is 1.5mg/ml
Standard Curve: allows one to accurately correlate absorbance in the spec to
protein concentration in the sample. Known standards must be used, such as BSA
diluted to various concentrations and mixed with colorimetric reagent.

Enzyme overview (function, activation energy)
Enzymes are proteins or RNA that are essential to cell function by increasing the rate of a
reaction. Highly specific; very tightly regulated by cells.
-Equilibrium: when the ratio of substrate to product remains the same. Equilibrium
constant can be calculated by dividing the forward (Ka) reaction rate by the reverse (Kb) reaction
rate. Enzymes do NOT affect equilibrium; reaction will reach equilibrium regardless of whether
enzyme is present or not. (Enzymes accelerate time it takes to reach equilibrium)
Enzymes work by stabilizing the transition state (TS) of a reaction and by decreasing the
activation energy.
Free Energy G: measure of difference in energy between substrates and products.
- G>0 = thermodynamically unfavorable, nonspontaneous (exothermic)
- G<0 = thermodynamically favorable, spontaneous (endothermic)
Active Site: 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 (occasionally reversible
covalent bonds).
-Active sites are highly conserved; changing single amino acid can affect
specificity or reaction rate.
-Cofactors (Metal) & Coenzymes (organic): help promote the reaction, stabilize
the transition state, or perform a mechanical function
-Conserved amino acids indicate important functions performed in the cell

Enzyme-substrate models
Lock & Key Model: substrate and the active site of the enzyme fit together like a key into
its lock (complementary). This model does not perfectly explain transition state stabilization.
Induced Fit Model: the binding of substrate induces conformational changes in the
enzyme which bring the substrates together and stabilizes the transition state.

Enzyme kinetics overview (rate, inhibitors)
Enzyme kinetics is the study of an enzymes rate.
Vmax: when the enzyme is saturated with substrate and catalyzes the reaction at its
maximum rate.
-Enzymes rate is its inherent velocity, and depends on the concentration of the
substrates, temperature, pH, and the presence of cofactors.
-Rate decreases as reaction equilibrium, substrate is depleted, product inhibits forward
reaction, or enzyme is inactivated.
Enzymes must be studied during INITIAL RATE PERIOD
Km, Michaelis Constant: is equal to the rates of breakdown of ES complex over its rate
of formation - measure of STABILITY of the ES complex.
-High Km = weak substrate binding
-Low Km = strong substrate binding
Inhibitors
Competitive inhibitors: bind to the same site on the enzyme that the substrates
bind (active site), thus competing with the substrate. Can be affected by substrate
concentrations (out-competed)
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 concentrations
Enzyme Assay- can be used to determine an enzymes rate. Measured by their activity (units)
-A unit of restriction enzyme is defined as the amount of enzyme required to cleave 1ug
of DNA in 1 hour in 50ul total volume
CDNB Assay: CDNB absorbs light @ 340nm when acted on by GST
-CDNB + GSH --(GST)--> 1-(S-glutathionyl)-2,4-dinitrobenzene
This assay can be used to track the kinetics of the reaction and the Km of the
substrates, and GST if often tagged to other proteins which allow researchers to
track the target protein by quantifying GST.
-Usually tracked with spectrophotmeter
Coupled Assay: the reaction of interest is linked to one that is measurable
-Pyruvate Kinase-Lactate Dehydrogenase Assay
Kinase Assay: used to study protein kinases, which phosphorylate other proteins. Can also be
used to study signaling pathways or screen enzyme inhibitors
Radiolabeled ATP is mixed with a protein kinase and a target can be phosphorylated with
the radioactive 32P from the ATP. Phosphorylated, radiolabeled proteins can be detected and
quantified by a scintillation counter or run on a gel and detected with autoradiography.
-Fig. on pg38

Chapter 5 Measurements, Concentrations, Dilutions, and Buffers
1 Dalton = 1 gram / mole
Milliliters to microliters conversion
1ml = 1000ul
Molarity, millimolar, micromolar, conversions between the three
1 M = 1 mole/L = 1000 millimoles/L
1mM = 1000 micromoles/L = 1000uM
1uM = 1000nM
Percent composition (v/v and w/v)
Weight/weight: both solute and solvent are expressed in the same physical mass units
Volume/volume: both the solute and the solvent are expressed in the same volume units
Weight/volume: the amount of solute is expressed in physical mass units, while the solution is
expressed in volume units.
Dilutions (MV = MV)
M1V1 = M2V2
Buffers be able to prepare a buffer as you did in lab or on the homework
PRACTICE PREPARING BUFFERS

Chapter 6 Laboratory Data, Experimental Design, and Research Ethics
Protocol writing: list of steps that should be followed to obtain the desired results.
-When designing a protocol, it is best to use your previous work or published protocols as
a guide.
-Everything needed to reproduce the experiment exactly should be included in the
protocol. Also note any deviations from standard protocol. Not allowed to refrain from
publishing entire protocol to harm competition.
Seminars, conferences, poster sessions
Overall goal of each of these is to present scientific data and research progress to
colleagues for feedback, networking, discussion, or to further advance the field.
Seminars: usually held at program or department level, and usually involves graduate
students and postdocs (some principal investigators PIs occasionally).
-Researcher presents anothers work, such as current paper of interest from a top
journal.
-Setting gives graduate students chance to practice reading papers and presenting
to others. Those in the audience are brought up to date on a specific area of
research
Invited Seminar Series: involves department or entire campus. Allows a large group of
scientists to see current research from an internal or external researcher, who may be given an
honorarium (payment).
-Allows researchers a chance to present their data to a group of colleagues,
interact with and teach students, network/get feedback, and promote their research
-May be used as part of an interview process for a position at the institution
Conferences/Symposiums: gatherings of experts in a field, sometimes once a year, which
features posters and invited lecturers. Allows scientists to present their research to colleagues,
learn new techniques, and discuss new theories and the direction of the field.
-Can be conferences regarding specific new techniques (crystallization) or
regarding a single topic (TB, HIV, Cancer)
-Lectures given by PIs
Poster Sessions: can be an on-campus display of ones research to other faculty and
fellow students. Can also be at a conference.
-Allows researchers who are not presenting to still show their work, and gives
grad students/postdocs the opportunity to show their data to others.
Grants- money to perform research, must apply for them. Apply to NIH or obtain private
donations.
Proposals- must have 3 goals. Greater chance of getting funded if researchers show that the work
is relevant, that current published works support their line of thought and that preliminary data
supports their hypothesis.
NIH review process- Each grant proposal is reviewed by a panel of scientists who give it a
score/percentile. If the score is high enough and the funds are available, the PI will receive the
money they requested.
-Some projects that use human/animal/certain pathogen as subjects will require
Institutional Review Board (IRB) approval before the grant is funded.
R01: most sought-after grant; can provide a lot of money for 3-5 years New investigators given
special consideration.

Research Publication
Relevant Title
Authors
Abstract- describes what was performed & the conclusions that were drawn.
Methods Section- (sometimes at very end of paper/written up separately in another
journal). Includes everything one would need to reproduce the experiments performed. Steps
should never be omitted. Only include deviations from standard protocols.
Results Section- contains all of the data and figures, and includes the specifics of the
paper. Usually very technical and detailed. Never should data be edited or falsified, or spliced
together from multiple images.
Discussion- where the authors analyze and discuss the data presented in the results
section, and bring all of the data together into a narrative. Usually contains most interesting and
exciting material in the paper as the authors conclusions, implications, and future direction.
References/Citations- also includes financial support list, gratitude to contributors who
are not authors, and a competing financial interests statement.
Impact Factor: total number of citations for each article in the following two years by
other research publications
Authorship Order: the PI of the lab who earned the grant that supported the work has the
final say in authorship. Usually:
-First Author: Graduate student or postdoc assigned to the project by the PI
-Second Author: Close collaborator on the project
-Middle Authors: Lab technicians or other research scientists who contributed to
the paper.
-Second to last Author: Supervising technical collaborator (possibly another PI)
-Last Author: PI of the lab, usually the corresponding author of the paper
Harvard Authorship Guidelines on pg.59
References/Citations: cite any statements made in the introduction to add validity to your
claims. Methods section may also include citations (for protocols), as may the discussion section.
Sources (even your own) should never be used word-for-word without quotes and citations.
Peer Review: where article is analyzed and criticized by scientists in the field. Usually
three anonymous peer reviewers; all reviewers questions must be answered, may sometimes
require further experiments. Final decision is often up to the journal editor, who can send the
article to a fourth reviewer for further comments.
Experimental Design review the steps
Scientific Method: Ask a question; identify the subject for experimental investigation;
evaluate the current state of knowledge by reading scientific literature on the subject, formulate a
hypothesis, select the biological system to be used, identify the variables and controls, test the
hypothesis, come to a conclusion. Also, possibly formulate a new hypothesis and test the new
hypothesis.
-If there is enough supporting (and no contradictory) evidence from many
experiments, a scientific theory can be proposed.

Variables: what is measured or varied in an experiment
Independent Variable: that which is manipulated (time, initial amounts, etc)
Dependent Variable: changes in response to the independent variable and is observed and
measured

Controls: Potential variables that must remain constant. Act as a baseline from which to measure
changes in the dependent variable.
-Good controls are the key to high quality scientific research.
Positive Control: are used as the normal test and should produce expected, measurable
results.
-Ensure the experiment worked as designed
Negative Control: should not be observed, and if they are seen it indicates that the sample
or reagents are contaminated.




Experimenter bias & Prevention
Experimenter bias, at the least, involves a researcher subconsciously biasing their data.
To prevent bias, scientists use good controls, statistically analyze data, report all data
(good & bad) and any manipulations thereof, undergo peer review process and experiment
duplication, use double-blind experiments, and hold to scientific rigor.
Scientific rigor: ensures the data and conclusions are supported across all tests, and
withstand any experiment they are put through.
Double Blind Experiment: tool used to prevent experimenter bias. In a double blind experiment,
the experimenter does not know which is the control group and which is the experimental group
-Are the key to combating anecdotal evidence.
-Not practical for day-to-day experiments in a research laboratory.
**Above all else, scientists must be 100% ethical, have absolutely no bias, and adhere to
scientific rigor when performing experiments.**

Chapter 19 - Cell Culture, Flow Cytometry, and Animal Research
A researcher who wishes to start a cell/animal project will: begin with full literature review,
followed by project design, bioinformatics analysis of the gene of interest, and then DNA
manipulation or protein purification. Next step would be to us cell culture to study in vitro, and
after possibly in vivo.
Model systems: system that will best fit experiment, often requires tradeoffs.
(advantages/disadvantages of each???)
Cell Culture Examples
Bacteria: inexpensive and efficient; proteins may not express, fold properly, or be
properly modified
S.cerevisiae (yeast): inexpensive and easy to work with; can be used to study eukaryotic
systems not found in bacteria, such as mitosis
Baculovirus expression vector system (BEVS): uses virus to insert genes into an insect
cell line. Cells can express large proteins in high amounts and contain proper post-translational
modifications. More expensive, take longer to grow (3-4 days).
Mammalian cell lines: effective because systems being studied are usually in their native
environment, resulting in proper expression and folding. Much more difficult to work with
require CO2 incubators, media must be changed every few days, and are not easily stored long-
term
Plants are also an alternative
Cell culture: an in vitro method that involves the incubation of biologically derived material in
an artificial physical and chemical environment, outside of a living organism.
Major disadvantage: artifacts may appearmechanisms or behaviors that only exist in a
cell culture (ex. abnormally high amount of hydrogen peroxide)
Cell culture Media (In special CO2 incubator): pH ~7.4, phenol red dye as pH indicator;
Possibly salts, vitamins, carbon sources, amino acids, proteins, growth factors=Fetal bovine
serum (FBS); Media must be changed frequently (~2 days); Require sterile techniques to prevent
bacterial contamination
Cells in culture may be modified or have genes inserted/removed

Transfection: the process of inserting DNA into eukaryotic cells (like transformation in bacteria);
resulting cells can have genes inserted or removed = pathways/disease states can be studied.
Eukaryotic cells can be transfected via CaCl2 or other cationic polymers; electroporation;
heat shocking; viruses (adeno-associated virus AAV) or retroviruses (HIV or lentivirus);
lipofection using vesicles called liposomes that fuse with the cell membrane;nanoparticles; gene
gun.
T-cell engineering: T-cell receptors are modified to target specific cell lines.
RNA interference: can be used to mediate sequence-specific degradation of a target mRNA, thus
shutting down translation/expression of a specific protein.
Microinjection: technique that involves adding or removing nuclear material (enucleation) or
injecting molecules into a cell with a small glass pipette.
Molecules can be inserted into cell
Nucleus can be completely removed/new one added.
Useful for plant manipulation as well
Immunohistochemistry/Immunocytochemistry (what do they tell you?): technique very similar to
a western blot, where antibodies recognize and bind to an antigen. In both techniques, antibodies
recognize a specific protein while it is still in its native cellular environment. Researchers can use
this technique to identify where proteins are found in the cell or tissue and track their expression
and localization.
Immunohistochemistry IHC tissues
Immunocytochemistry ICC cells
Pulse-Chase (purpose): Used to determine metabolic activity by tracking radiolabeled amino acid
through a cell. Cell is exposed to radioactive molecule (Pulse), and after a few minutes exposed
to a non-radioactive molecule (Chase); this radioactivity can be tracked throughout the cell; can
follow metabolite, protein, or nucleotide through cellular compartments.
Electrophysiology & Patch Clamp (purporse): Electrophysiology is the study of the electrical
properties of a cell
Microelectrodes attached to an organ; Patch clamp allows scientist to suck up a small
section of the plasma membrane in a pipette, which contains an electrodxe that measures changes
in current as ion channels in the cell open & close.
Readings are recorded on an oscilloscope.
Cell sorting FACS/flow cytometry purpose, data analysis from histogram
Cell Sorting: the isolation in bulk of individual cells or populations of particular cells
from a mixed population.
Bacteria & Eukaryotes can be selected based on ability to survive:
-Eukryotes transfected to make them resistant to infection, cytotoxins, or
antibiotics
Cells can also be separated on basis of physical properties (RBC from WBC via
centrifugation).
-Also separation by surface charge, electrophoresis, affinity chromatography,
agglutination, or phase partitioning.
Flow Cytometry (for eukaryotic cells): allows a researcher to separate cells based on size,
health, or other properties such as cell stage or number of chromosomes.
-Technique uses a vibrating nozzle to produce small droplets from a cell sample
that contains several cells. The droplets pass through a laser that detects various
cell properties. A charge can be added to a drop, which allows a deflector to
divert the sample to several collection tubes. Samples often passed through a flow
cytometer various times to ensure proper sorting. Figure on pg227
-Forward scatter = size / side scatter = granularity (health)
-Forward & side scatter can be recorded and presented as histogram. Individually,
the histograms represent the size and health of the cells, and together the entire
cell population can be analyzed based on both properties = Scatter plot
Fluorescence-activated Cell Sorting (FACS): improvement to flow cytometry; allows one
to sort cells based on light scattering and fluorescence.
-Offers researchers a wide range of properties upon which to sort cells
-Cell-binding fluorescent dyes can be very specific to cell types or properties (also
to DNA, organelles, or proteins. GFP-tagged proteins can also be detected via
FACS
-FACS can be used diagnostically to determine cell ploidy or
immunophenotyping to screen cell surface markers (such as CD markers in the
Luminex Assay) to determine a patients disease state.
-Scatter plot figure on pg.228

In situ Organ perfusion: technique involves infusing a compound (like formaldehyde and
sucrose) through a fine hollow needle inserted into the artery carrying blood to the organ, and
performing subsequent analysis on the blood being transported from that organ.
-Can be used to study any organ (or interaction of a drug with an organ)
-Organ is removed from animal, maintained at constant temperature and pH in apparatus,
and fluid is passed through the organ by gravity/pump that resembles heart flow.
Whole-body autoradiography (WBA): Entire slices of an animal are made. Animal is fed a radio-
labeled drug, killed, frozen, and then sliced. Whole-body autoradiography is then used to trace
the localization of the drug in the slice to determine where it takes effect.
-Can be used to track tumor growth, drug localization, or tumor/drug interactions
Animal Studies (advantages and disadvantages): used when experiments require more than in
vitro cell cultures to obtain data.
C. elegans (roundworm), Drosophila (fruit fly), & zebrafish: commonly used due to their
simplicity
-C. elegans has same basic organs and body plan as all animals in only 959
(hermaphrodite) or 1031 (male) cells, and is transparent (visualization). Nervous
system has been completely mapped.
-C. elegans gene expression easy to disrupt via RNA interference
-Drosophila very useful for studying genetics
-Zebrafish very useful for studying development
Mice, rats, guinea pigs: ease of use, low cost, large litters
-Mice genome has been sequenced; 99% gene homology with humans; similar
gene order as well.
-Mouse techniques perfected; genome easy to manipulate
Animals bred specifically for experimental purposes under scrutiny of Institutional
Animal Care and Use Committee (IACUC)
-Animals only used if in vitro models not suitable for experiments.
Disadvantages: animals dont always produce human-like patterns of pathology, or
respond in the same way to a particular treatment.
-Animal models = 66% prediction of human toxicity
-Rodents even less predictive
Transgenic animals: have manipulated genomes or carry genes from another species. Process
involves injecting the gene or interest into an animal zygote and implanting this embryo into a
surrogate female.
-Animals designed to mimic a human disease state
NOG Mouse: severely immunodeficient mouse that has been designed to lack T cells, B
cells, NK cells, and has defective macrophage, dendritic cells, and complement functions.
RNA interference (RNAi): can be used in vivo. siRNA injected into a living animal
instead of a cell culture. Can be used to study the role of specific genes in animals, as silencing
expression can cause observable changes.
Cloning: process of creating an exact copy of a living organism. Dolly the sheep in 1996;
Mouse in 2008; Pyrenean ibex in 2009 (had been extinct), survived briefly.
-Original cloning: involved removal of the nucleus from donor, transfer of a new
nucleus from donor, egg then transplanted into surrogate mother
-New process: donor cells transferred to cell culture, genetically engineered into
pluripotent stem cells by transfection of certain genes or demethylation of DNA,
and the cells are then injected into a donor egg and surrogate mother. (This
process is EXTREMELY INEFFICIENT)
Knock-out: involves deleting a gene; extremely useful in determining that genes function in the
cell.
Knock-in: involves inserting a gene
Knock-down: involves partial gene disruption; may be needed if a gene is embryonic
lethal if missing.
-All three techniques begin with basic DNA manipulation (PCR, cloning, transformation,
transfection, cell culture) and use some advanced techniques to knock in or knock out a specific
gene.
-Procedure: Mouse embryonic stem cells manipulated via homologous recombination so
that they gain/lose gene of interest. Cells then selected for and injected into a mouse blastocyst,
which is injected into a surrogate mother. Resulting mouse is a Chimera (contains cells from two
different mice).
-Crossing chimeras will result in the production of a knockout mouse that lacks
the gene of interest.
Cre-Lox mouse (Conditional knockout): involves knocking out a target gene only in a specific
cell type. Use Cre recombinase (from a bacteriophage) to cause recombination between two
LoxP sites (a specific sequence of DNA). Genes are turned off via inversion or complete
removal.
First, Cre mouse is produced that contains Cre recombinase in its genome, with a cell-
specific upstream transcription factor binding site. It is then cross with a LoxP mouse,
which has the gene of interest flanked by LoxP sites. The resulting Cre-LoxP mouse will
express Cre recombinase only in cells that contain the correct transcription factor. Cre
recombinase will seek out the LoxP sites and remove the gene. A reporter system (GFP,
LacZ) can be used to verify that the gene has been turned off
Optogenetics: Algae express protein channelrhodopsin embedded in the cell membrane that
uncoils and opens up channels in response to light, which turns on flagella (in the algae). This
gene and a specific promoter are inserted into a retrovirus and injected into the brain of a mouse;
the virus then inserts the new gene into every neuron in the vicinity. Because of the promoter the
gene will only be expressed in specific neurons. When light strikes the neuron, channelrhodopsin
opens and the neuron fires.
-Other rhodopsin variants can be used to actually inhibit neuron firing
NOTES FROM LECTURE
BONUS: what are HeLa cells? Cervical cancer cell line taken from Henrietta
Lacks (?)

Chapter 20 - Bioinformatics and Computer-Based Research
Bioinformatics overview: A large amount of molecular biology research today relies on some
form of computational analysis
The computational branch of molecular biology is called bioinformatics
-This branch ties together molecular biology, physics, chemistry, biochemistry,
computer science, math, statistics, informatics, and artificial intelligence.
-Uses algorithms and databases to analyze DNA sequences, gene expression,
genomes, protein structures, protein interactions, and drug interactions
-In silico: techniques performed in a computer
Genome sequencing techniques: roots of bioinformatics is in genome sequencing of the 1970s
RNA genome of bacteriophage MS2 sequenced in 1976
Frederick Sanger sequences genome of bacteriophage -X174 (had previously sequenced
insulin in 1955
Haemophilus influenza: 1.8 million bp; first non-viral genome to be sequenced (1995);
scientists at Johns Hopkins University
Human Genome Project: started in 1990 by James Watson at NIH, funded by Dept. of Energy.
Taken over by Francis Collins in 1992.
Goal was to sequence 3 billion chemical base pairs that make up human DNA, identify
20,000-25,000 human genes, and store all this information in databases. Also, to improve DNA
data analysis, transfer related technologies to private sector, and apply knowledge gained to other
fields.
Cost $3 billion in public funds, $300 million by Celera- $100 per bp
End Result: near-complete sequence of the human genome stored in a computer database
accessible to all. Only regions not sequenced were centromeres and telomeres = highly repetitive
and difficult to sequence by current methods.
Two approaches used: chromosome walking & shotgun sequencing. Together, two
approaches helped to produce the complete human genome in the early 2000s
Chromosome walking: genomic DNA carried in a clone is sequenced, and a fragment of
this sequence is used as a probe to isolate the next clone, which is sequenced next. Only 500-
1000 bp of DNA could be sequenced at a time (very challenging)
Shotgun sequencing: put into practice in the human genome by Craig Venter. Human
genome is mechanically sheared, polished to add adenines to each piece of DNA, incorporated
into plasmids via TA cloning, and sequenced using plasmid-specific primer. Sequenced
fragments are then pieced back together on a computer.
Human genome laid the foundation for using genomic info for drug discovery, led to new
field of study systems biology: the study of entire biological systems and their interactions
between components.
Genomics: the study of an organisms entire genome.
NCBI: National Center for Biotechnology Information National resource for molecular
biology information, NCBI creates public databases, conducts research in computational biology,
develops software tools for analyzing genome data, and disseminates biomedical information
all for the better understanding of molecular processes affecting human health and disease
GenBank: database that contains DNA sequences amounting to billions of base pairs and DNA
from thousands of different organisms, from viruses to humans. Journals expect researchers to
submit their DNA and protein sequences to GenBank prior to publication.
PubMed: searchable database of thousands of scientific journals. First place that one should visit
when looking for peer-reviewed scientific data on a topic, or when performing a full literature
review for a research project.
BLAST: Basic Local Alignment Search Tool; tool for comparing any sequence with which you
are working to the entire GenBank database.
Can identify what gene the sequence is, or any similar genes in the database.
Also useful for finding similar sequences among various species
-If two sequences are ~30% identical or more (whether within a species or across
species), it usually indicates they have a similar function
Sequence Alignment: sequences from blast search are further analyzed by looking for
conserved nucleotides (BLASTn - nucleotide) or amino acids (BLASTp - protein) across species
-Alignment provides information useful for evolutionary analysis or can be used
to determine which amino acids play an important role in a proteins function.
Identical amino acids across species usually indicate that they serve an important
function as they have been conserved since the last common ancestor.
Phylogenetic Tree: can be used to display sequence data visually and determine relationships
between species. Can be created with DNA or protein sequences.
ExPASy: Expert Protein Analysis System site contains tools and software used to analyze
DNA and protein sequences. Some of the tools allow for protein identification and
characterization (Mw, pI), DNA to protein conversion, similarity searches, post-translational
modification and topology prediction, and more..
Sequence analysis pI, Mw, regions of homology, percent identity, phylogenetic tree, etc.
Secondary structure analysis: Most basic technique for secondary structure prediction is to use
alignments with known sequences or structures.
Chou and Fasman (1974): their techniques can be used when no homologous structures
exist. Their analysis of proteins showed that certain amino acids were more likely to be involved
in alpha-helices, B-sheets, and turns than others. Approx. 50% accurate
Garnier-Osguthrope-Robson (GOR): analyzes a larger segment of amino acids (17 AAs
total) to predict protein structures. Approx 75% accuracy.
Ultimate goal of bioinformatics (with regards to protein structure) is to be able to predict
exact tertiary structures from the primary sequence.

Ramachandran Plot: this plot analyzes the phi and psi angles around the alpha-carbon of
an amino acid. Certain combinations of phi and psi tend to be found in specific secondary
structures, and some AAs are limited to certain phi and psi angles by steric hindrances.
-Alpha-helix: phi = -60 and psi = -50
-B-sheet: typically have positive psi.
-Plots useful for predicting secondary structures, but also used as a refinement
tool to ensure all angles are within acceptable limits when solving proteins
structures.
Helical Wheel: used to predict an alpha-helix. Proteins can be analyzed to look for
pattern of alternating residues to not only discover new alpha-helices, but predict their location
and possible function in the overall protein structure.
-Globular proteins and leucine zippers often have alpha-helices with alternating
hydrophobic and hydrophilic residues. It is more favorable for hydrophobic AAs
in a protein to be buried internally away from the solvent
Hydrophobicity Plot: AAs can be polar or nonpolar; this plot assigns a number to each
amino acid based on how hydrophobic or hydrophilic it is (Fig on pg240). A sequence can be
analyzed to look for hydrophobic or hydrophilic stretches of amino acids.
-Hydrophobicity information can be useful in predicting transmembrane proteins
which have long stretches (~20) of hydrophobic AAs followed by stretches of
hydrophilic amino acids (Figure on pg241). Hydrophobic AAs are buried in the
plasma membrane, while the hydrophilic AAs are exposed to the solvent on
inside/outside of cell.
Disorder Plot: another type of secondary structure analysis, which looks for regions of
high movement or disorder. Often, such a stretch of AAs are important in protein function,
ligand binding, protein-protein interactions, and protein activation.
Protein Data Bank (PDB): once the tertiary structure of a protein is solved, its coordinates
are deposited into the Research Collaboratory for Structural Bioinformatics Protein Data Bank.
These structures are free and accessible from any computer. Currently contains over 10k protein
structures, including small molecules, DNA, RNA, and more.

Structural modeling: involves taking unknown sequences and predicting tertiary structures, or
taking known structures and modeling protein activation, protein-protein interactions, ligand-
binding, and much more.
-Accurately predicting tertiary structures is very difficult because of large number of
structures possible from even a handful of AAs
Molecular Mechanics: most common technique for accurately predicting tertiary
structures from scratch (ab initio).
-This technique uses the atomic nuclei and force fields to predict structures and
interactions.
-Fast; fairly accurate; able to handle millions of atoms
-Still not a perfect representation of what is occurring at molecular level.
-Quantum Mechanics: takes into account the electrons of the atoms and wave-
particle duality, along with other complex properties. Produced structures are very
accurate, but require a lot of computing power, and are still size-limited.
Energy minimization/Simulated annealing: Once tertiary structure is solved, it is further
optimized through this technique. Temperature of molecule in the computer is increased, whence
it will begin to vibrate greatly, and as it cools it will hopefully find the energy minimum, or the
most favorable (and likely) structure.
Molecular Dynamics: another aspect of structural modeling that allows one to model
molecular interactions, like a protein with a binding partner. (protein with binding partner,
myoglobin with oxygen, etc..)
Virtual screening (Structure-based design): was an answer to lack of diversity produced by
medicinal chemistry and the high cost of high-throughput screening. It is an efficient way to
screen thousands of potential protein inhibitors on a computer.
-Cost effective; reduces false-positives; increases the number and diversity of potential
inhibitors; helps to find new classes of drugs.
-Take a known structure, mark the site to which a potential inhibitor should bind, search a
database for drugs that would bind to the site, and score possible hit.
-Once potential inhibitor is identified, it can be modified, synthesized, or purchased and
tested in vitro to see if it inhibits the target.
Proteomics: the study of all of the expressed proteins in an organism, including isoforms and
modifications. Purpose is to produce comprehensive protein databases of gene function and help
to lay the groundwork, along with genomics, for systems biology (studying entire organism).

NOTES FROM LECTURE:
Carsonella ruddii (proteobacteria) 159,662 (182 genes) = Smallest genome weve
found
2.86/3.1 Gb sequenced (92%) of Human Genome
Benlysta (treatment for Lupus) one of the first drugs to come out of the genomics
revolution.
If two proteins have 30% identity/homology = very good chance both have same
function
RecA (e.coli) & RAD51 (Humans) only have 15% homology but structurally there is a
lot of homology; both have very similar functions
-X174 (5,368 bp) infects E. coli
Long stretches (~20) of nonpolar/hydrophobic AAs can indicate that the protein is
transmembrane
NMR can capture movement; Although X-ray crystallography is usually better, it cannot
capture movement
Issue with virtual screening is that the structure of the drug targets have not been resolved
and are also often times transmembrane proteins
Dorzolamide (1995), an anti-glaucoma drug that targets carbonic anhydrase, is the result
of structure-based drug design
Creating life 2014 synthesized functional yeast chromosome from scratch (272kb)



Chapter 21 - Mass Spectrometry, Microscopy, NMR, and Crystallography
Mass spectrometry (MS): allows one to determine the mass of a molecule with extreme
accuracy. Useful for identifying, verifying, and quantitating metabolites, proteins and peptides,
oligos, and much more.
In MS, molecules are converted into a charged, gaseous state and analyzed by a detector.
Gaseous ions are separated according to their mass-to-charge ratio (m/z) and the output is a plot
of the relative abundance of the ions at each m/z ratio.
Electrospray Ionization: used to analyze larger molecules (like proteins). Sample is
sprayed with a charged solvent and detected.
Liquid Chromatography Mass Spectrometry (LC-MS): injects liquid directly into the
mass analyzer.
Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF): uses a laser to
gently ionize large molecules. Smaller molecules diffuse more quickly, and reach the detector
first. The time it takes for the molecule to reach the detector is measured and converted into
molecular weight.
-Mass spectrometers can detect isotopes if their resolution is high enough = better mass
accuracy achieved
-MS can be used to identify proteins: proteins are digested and subjected to one of
various types of MS. The resulting spectra will give the Mws of any fragments in the mixture,
which can be compared to a table of known Mws of protein sequences.

Monoisotopic mass vs. average mass
Inverted Microscope: has optics below the stage, unlike a traditional microscope (with the optics
on top of the stage).
-Especially useful for cell cultures since cells tend to be on the bottom of tissue culture
flasks
Fluorescence Microscopy: allows researchers to view aspects of the cell not visible with a
normal microscope
-Molecule is labeled with GFP or a dye, which is excited by a specific wavelength to
fluoresce.
-Fluorescent Abs or chemicals that target a specific cell structure can be used, or genes
that encode proteins that are tagged with GFP can be transfected into cells. Proteins can be
tracked as they move throughout cell
Confocal microscopy: similar to fluorescence microscopy, but uses laser to greatly improve the
image quality and resolution.
Electron microscopy: allows researchers to view samples at up to 10 million fold magnification.
Electrons, which have very small wavelengths, are focused to resolve an image.
Transmission Electron Microscopy (TEM): electron beam goes through the sample.
Higher magnification.
Scanning Electron Microscopy (SEM): beam bounces off of a gold-plated sample. More
useful as it only reveals surface of a sample
Cryo-electron Microscopy (Cryo-EM): many copies of a sample are frozen and placed under an
EM. Hundreds of thousands of pictures are taken of the sample from various angles, and these
images are reassembled into a single image.
-Has reached 4 Angstrom resolution (enough to trace a polypeptide chain.
-Resolution not as high as NMR and X-Ray crystallography, but works well in
conjunction with these other techniques to provide low resolution information.
Rotary Shadowing: technique allows one to view individual proteins and macromolecules
without the need for advanced procedures, but at much lower resolution.
Atomic force microscopy: technique developed to visualize small molecules and proteins.
Device is similar to a tuning fork that vibrates near molecules, causing a vibration that can be
measured.
-Technique has been used to visualize molecular structures and myosin walking along
actin
Circular Dichroism (CD): allows one to get an idea of the secondary structure features of a
protein. CD measures differences in the absorption of left-handed polarized light vs. right-hand
polarized light, which arise due to structural asymmetry in alpha-helices and B-sheets.
-Not adequate for determining tertiary structure

NMR: used to determine tertiary structure. Technique utilizes very strong magnetic fields to
determine the relative positions of certain atoms in the protein that have nonzero spins and thus
magnetic and angular momentum. Interactions between these various atoms and their neighbors
can be measured in the presence of the magnetic field and the atomic structure can be determined
-Allows researchers to study molecules directly in solution without the need for a crystal,
which produces more mobile, flexible structures. (Advantage over crystallography)
-NMR structures are very accurate. Limited to 30kDa in size.
X-Ray Crystallography: can be performed on anything, so long as a crystal of the molecule is
available. Crystals generally produced by purifying the protein of interest via chromatography,
concentrating it to 10mg/ml, and placing it over a reservoir of precipitant that allows for the
water in the droplet to slowly evaporate, promoting crystal formation.
Asymmetric unit: most basic unit for a crystal.
Unit cell: the smallest unit of repeat within a crystal.
-Crystal is placed in front of a very powerful X-ray beam. Rays generally travel through,
but can also interact with electrons of the protein and scatter. The diffraction pattern is collected
on film/by camera. Crystal is also rotated to collect data from all sides to determine structure.
Space group: orientation of the crystals subunits; broken down into 7 major groups
based on symmetry: cubic, hexagonal, rhombohedral, tetragonal, orthorhombic, monoclinic, and
triclinic + 230 subclasses. Only 32 protein crystal classes
Constructive interference (in phase) vs. Destructive interference (out of phase): incident
X-ray strikes a specific electron and the deflected beam results in a diffraction spot vs. no
beam/electron interaction = no spot detected. Data is then used to create an electron density map
Imaginary space: created by the resulting diffraction image; is unfocused; equivalent to
the light that strikes the lens of your eye before being focused. Diffraction spot intensity provides
information regarding electron density in a protein.
Fourier Transform: converts imaginary space to real space. Data from hundreds of
images is integrated into single data set = structure can be determined.
Phase problem: scientist must determine the phase at which the light wave struck the
crystal to solve the structure. Majority of image info is carried in the phase, not intensity.
-Molecular replacement: known structure used to solve unknown structure.
-Multiple isomorphous replacement: heavy metals are soaked in the crystal and
their diffraction is located in the data
-Anomalous dispersion: (requires synchrotron) selenomethionine or other
molecules are used to measure differences in the crystals diffraction at 180
degrees.
-END RESULT: electron density map which shows where X-rays interacted with
electrons. Protein structures can be built into the electron density map and refined.