BE176 – Cellular and Molecular Biology Notes Module 2

Topic 1: Molecular Tools for Studying Genes and Gene

Activity
1.1 Molecular Separations
 Mixtures of proteins or nucleic acids are generated during the course of molecular
biological procedures
o A protein may need to be purified from a crude cellular extract
o A particular nucleic acid molecule made in a reaction needs to be purified

1.2 Gel Electrophoresis

 Used to separate different species of:
o Nucleic acids
o Protein

1.2.1 DNA Separation by Agarose Gel Electrophoresis

 Melted agarose is poured into a form equipped with removable comb
 Comb “teeth” form slots in the solidified agarose
 DNA samples are placed in the slots
 An electric current is run through the gel at a neutral pH
 DNA is negatively charged due to phosphates in its backbone and moves to anode, the
positive pole
o Small DNA pieces have little frictional drag so move rapidly
o Large DNAs have more frictional drag so their mobility is slower
o Result distributes DNA according to size
 Largest near the top
 Smallest near the bottom
o DNA is stained with fluorescent dye

1
BE176 – Cellular and Molecular Biology Notes Module 2

1.2.2 DNA Size Estimation

 Comparison with standards permits size estimation
 Mobility of fragments are plotted v. log of modular weight (or number of base pairs)
 Electrophoresis of unknown DNA in parallel with standard fragments permits size
estimation
 Same principles apply to RNA separation

1.2.3 Electrophoresis of Large DNA

 Special techniques are required for DNA fragments larger than about 1 kB
 Instead of constant current, alternate long pulse-field gel electrophoresis (PFGE)

1.2.4 Protein Gel Electrophoresis

 Separation of proteins is done using a gel made of polyacrylamide
o PAGE = polyacrylamide gel electrophoresis
o Treat proteins to denature subunits with detergent such as SDS
 SDS coats polypeptides with negative charges so all move to anode
 Masks natural charges of protein subunits so all move relative to mass not
charge
o As with DNA smaller proteins move faster toward the anode

2
BE176 – Cellular and Molecular Biology Notes Module 2

1.2.5 Summary
 DNAs, RNAs, and proteins of various masses can be separated by gel electrophoresis
 Most common gel used in nucleic acid electrophoresis is agarose
 Polyacrylamide is usually used in protein electrophoresis
 SDS-PAGE is used to separate polypeptides according to their masses

1.3 Two-Dimensional Gel Electrophoresis

 While SDS-PAGE gives a good resolution of polypeptides, some mixtures are so
complex that additional resolution is needed
 Two-dimensional gel electrophoresis can be done
o (no SDS) uses 2 consecutive gels
o Sequential gels with first a pH separation then separate in a polyacrylamide gel

1.3.1 A Simple 2D Method

 Run samples in 2 gels
o First dimension separates using one concentration of polyacrylamide at one pH
o Second dimension uses different concentration of polyacrylamide and pH
o Proteins move differently at different pH values without SDS and at different
arylamide concentrations

1.3.2 Two-Dimensional Gel Electrophoresis Details

 Isoelectric focusing gel
o Two process method
o Mixture of proteins electrophoresed through gel in a narrow tube containing a pH
gradient
 Negatively charged protein moves to its isoelectric point at which it is no
longer charged
 Tube gel is removed and used as the sample in the second process
 Standard SDS-PAGE
o Tube gel is removed and used as the sample at the top of a standard
polyacrylamide gel

3
BE176 – Cellular and Molecular Biology Notes Module 2

o Proteins partially resolved by isoelectric focusing are further resolved according

to size
 When used to compare complex mixtures of proteins prepared under two different
conditions, even subtle differences are visible

1.4 Ion-Exchange Chromatography

 Chromatography – originally referred to the pattern seen after separating colored
substances on paper
 Ion-exchange chromatography uses a resin to separate substances by charge
 Especially useful for proteins
 Resin is placed in a column and sample loaded onto the column material

1.4.1 Separation by Ion-Exchange Chromatography

 Once the sample is loaded buffer is passed over the resin + sample
 As ionic strength of elution buffer increases, samples of solution flowing through the
column are collected
 Samples are tested for the presence of the protein of interest

4
BE176 – Cellular and Molecular Biology Notes Module 2

1.5 Gel Filtration Chromatography

 Protein size is a valuable property that can be used as a basis of physical separation
 Gel filtration uses columns filled with porous resins that let in smaller substances,
exclude larger ones
 Larger substances travel faster through the column

1.6 Affinity Chromatography

 Resin contains a substance to which the molecule of interest has a strong and specific
affinity
 The molecule binds to a column resin coupled to the affinity reagent
o Molecule of interest is retained
o Most other molecules flow through without binding
o The molecule of interest is eluted from the column using a specific solution that
disrupts the specific binding

1.6.1 Labeled Tracers

 Labeled – radioactive
 Radioactive tracers allow vanishingly small quantities of substances to be detected
 Molecular biology experiments typically require detection of extremely small amounts of
a particular substance

1.6.2 Autoradiography
 A means of detecting radioactive compounds with a photographic emulsion
o Preferred emulsion is x-ray film
o DNA is separated on a gel and radiolabeled

5
BE176 – Cellular and Molecular Biology Notes Module 2

o Gel is placed in contact with x-ray film for hours or days

o Radioactive emissions from the labeled DNA expose the film
o Developed film shows dark bands

1.6.3 Autoradiography Analysis

 Relative quantity of radioactivity can be assessed looking at the developed film
 More precise measurements are made using densitometer
o Area under peaks on a tracing by a scanner
o Proportional to darkness of the bands or autoradiogram

1.6.4 Phosphorimaging
 This technique is more accurate in quantifying amount of radioactivity in a substance
o Response to radioactivity is much more linear

6
BE176 – Cellular and Molecular Biology Notes Module 2

o Place gel with radioactive bands in contact with a phosphorimager plate

o Plate absorbs beta-electrons that excite molecules on the plate which remain
excited until plate is scanned
o Molecular excitation is monitored by a detector

1.6.5 Liquid Scintillation Counting

 Radioactive emissions from a sample create photons of visible light are detected by a
photomultiplier tubs in the process of liquid scintillation counting
o Remove the radioactive material (band from gel) to a vial containing scintillation
fluid
o Fluid contains a fluor that fluoresces when hit with radioactive emissions
o Acts to convert invisible radioactivity into visible light

1.6.6 Nonradioactive Tracers

 Newer nonradioactive tracers now rival older radioactive tracers in sensitivity
 These tracers do not have hazards
o Health exposure
o Handling
o Disposal
 Increased sensitivity is from use of a multiplier effect of an enzyme that is coupled to
probe for molecule of interest

1.6.7 Detecting Nucleic Acids with a Nonradioactive Probe

7
BE176 – Cellular and Molecular Biology Notes Module 2

1.7 Southern Blots

1.7.1 Using Nucleic Acid Hybridization
 Hybridization – the ability of one single-stranded nucleic acid to form a double helix with
another single strand of complementary base sequence

1.7.2 Southern Blots: Identifying Specific DNA Fragments

 Digests of genomic DNA are separated on agarose gel
 The separated pieces are transferred to filter by diffusion, or more recently by
electrophoresing the bands onto the filter
 Filter is treated with alkali to denature the DNA, resulting ssDNA binds to the filter
 Probe the filter using labeled cDNA
 Probe cDNA hybridizes and a band is generated corresponding to the DNA fragment of
interest
 Visualize bands with x-ray film or autoradiography
 Multiple bands can lead to several interpretation
o Multiple genes
o Several restriction site in the gene

1.7.3 DNA Fingerprinting and DNA Typing

 Southern blots are used in forensic labs to identify individuals from DNA-containing
materials
 Minisatellite DNA
o A sequence of bases repeated several times

8
BE176 – Cellular and Molecular Biology Notes Module 2

o Also referred to as DNA fingerprint

o Individuals differ in the pattern of repeats of the basic sequence
o Difference is large enough that 2 people have only a remote chance of having
exactly the same pattern
 General steps
o Cut the DNA under study with restriction enzyme
 Ideally cut on either side of minisatellite but not inside
o Run digest on a gel and blot
o Probe with labeled minisatellite DNA and imaged
 Real samples result in very complex patterns

1.7.4 Forensic Uses of DNA Fingerprinting and DNA Typing

 While people have different DNA fingerprints, parts of the pattern are inherited in a
Mendelian fashion
o Can be used to establish parentage
o Potential to identify criminals
o Remove innocent people from suspicion
 Actual pattern has so many bands they can smear together indistinguishably

9
BE176 – Cellular and Molecular Biology Notes Module 2

o Forensics uses probes for just a single locus

o Set of probes gives a set of simple patterns

1.7.5 In Situ Hybridization: Locating Genes in Chromosomes

 Labeled probes can be used to hybridize to chromosomes and reveal which chromosome
contains the gene of interest
o Spread chromosomes from a cell
o Partially denature DNA creating single-stranded regions to hybridize to labeled
probe
o Stain chromosomes and detect presence of label on particular chromosome
 Probe can be detected with a fluorescent antibody in a technique called fluorescence in
situ hybridization (FISH)

1.8 Northern Blots

 Obtain RA from different tissues
 Run RNA on agarose gel and blot to membrane
 Hybridize to a labeled cDNA probe
 Can indicate the abundance of the transcript
 Densitometer – can quantify the abundance of RNA

10
BE176 – Cellular and Molecular Biology Notes Module 2

1.9 Immunoblots (Western Blot)

 Use a similar process to Southern blots
 Electrophoresis of proteins
 Blot the proteins from the gel to a membrane
 Detect the protein using antibody or antiserum to the target protein
 Labeled secondary antibody is used to bind the first antibody and increase the signal

1.10 DNA Sequencing

 Sanger, Maxam, Gilbert developed 2 methods for determining the exact base sequence of
a cloned piece of DNA
 Modern DNA sequencing is based on the Sanger method
1.10.1 Sanger Manual Sequencing
 Uses dideoxynucleotides to terminate DNA synthesis
 Process yields a series of DNA fragments whose size is measured by electrophoresis
 Last base in each fragment is known as that dideoxynucleotide was used to terminate the
reaction
 Ordering the fragments by size tells the base sequence of the DNA

11
BE176 – Cellular and Molecular Biology Notes Module 2

1.11 Automated DNA Sequencing

 Manual sequencing is powerful but slow
 Automated sequencing uses dideoxynucleotides tagged with different fluorescent
molecules
o Products of each dideoxynucleotides will fluoresce a different color
o Four reactions are completed, then mixed together and run out on one lane of a
gel

12
BE176 – Cellular and Molecular Biology Notes Module 2

1.11.1 Mapping Experiment

13
BE176 – Cellular and Molecular Biology Notes Module 2

1.11.2 Using Restriction Mapping with an Unknown DNA Sample

1.11.3 Mapping the Unknown

14
BE176 – Cellular and Molecular Biology Notes Module 2

1.11.4 Southern Blots and Restriction Mapping

1.12 Summary
 Physical map tells about the spatial arrangement of physical “landmarks” such as
restriction sites
o In restriction mapping cut the DNA in question with 2 or more restriction
enzymes in separate reactions
o Measure the sizes of the resulting fragments
o Cut each with another restriction enzyme and measure size of subfragments by gel
electrophoresis
 Sizes permit location of some restriction sites relative to others
 Improve process by Southern blotting fragments and hybridizing them to labeled
fragments from another restriction enzyme to reveal overlaps

1.12.1 Protein Engineering with Cloned Genes: Site-Directed Mutagenesis

 Cloned genes permit biochemical microsurgery on proteins
o Specific bases in a gene may be changed

15
BE176 – Cellular and Molecular Biology Notes Module 2

o Amino acids at specific sites in the protein product may also be altered
o Effects of those changes on protein function can be observed
 Might investigate the role of phenolic group on tyrosine compared to phenylalanine

 Can introduce changes using cloned gene that can alter amino acid sequence of protein
products
 Mutagenized DNA can be made with
o Double-stranded DNA
o Two complementary mutagenic primers
o PCR
 Digest the PCR Product to remove wild-type DNA
 Cells can be transformed with mutagenized DNA

1.13 Knockouts
 Probing structures and activities of genes does not answer questions about the role of the
gene in the life of the organism

16
BE176 – Cellular and Molecular Biology Notes Module 2

 Targeted disruption of genes is now possible in several organisms

 Knockout mice
o Products whose genes are disrupted
1.13.1 Stage 1 of the Knockout Mouse
 Cloned DNA containing the mouse gene to be knocked out is interrupted with another
gene that confers resistance to neomycin
 A thymidine kinase gene is placed outside the target gene
 Mix engineered mouse DNA with stem cells so interrupted gene will find way into
nucleus and homologous recombination with altered gene and resident, intact gene
 These event are rare, many cells will need to be screened using the introduced genes

1.13.2 Stage 2 of the Knockout Mouse

 Introduce the interrupted gene into a whole mouse
 Inject engineered cells into a mouse blastocyst

17
BE176 – Cellular and Molecular Biology Notes Module 2

 Embryo into a surrogate mother who gives birth to chimeric mouse with patchy coat
 True heterozygote results when chimera mates with a black mouse to produce brown
mice, half of which will have interrupted gene

1.13.3 Knockout Results

 Phenotype may not be obvious in the progeny, but still instructive
 Other cases can be lethal with the mice dying before birth
 Intermediate effects are also common and may require monitoring during the life of the
mouse

18
BE176 – Cellular and Molecular Biology Notes Module 2

Topic 2: Biological Model Systems

2.1 Important Features of Any Experiment
 Given a specific question, one can then come up with answers to the following three key
properties of the experiment
o What biological system?
 E.g. Do I look at human cell lines, a mouse, yeast, etc.?
o What perturbation/treatment conditions?
 E.g. What compounds should I apply to the system to elicit a relevant
response?
o What measurements?
 E.g. What transcripts do I need to look at, and/or do I need to look at
protein levels instead?
 Often (but not always), if you can’t design an experiment that only has a handful of
conditions and measurements, results may be difficult to interpret
o Usually, the question is too complex or not significant
o Exceptions are screening based studies, but those are also typically have a specific
question of interest

2.2 What is a model?

2.2.1 Model Definition
 A simplified/convenient version of a complex/inconvenient entity
 The model captures the key characteristics of the entity needed for addressing the task at
hand
 Why do we use models for biological research?
o Directly studying the target (human disease) is difficult
2.2.2 Types of Models
 Mouse Model
 Cell line Model
 Computational Model

19
BE176 – Cellular and Molecular Biology Notes Module 2

2.3 Whither Model Organism Research

 “Over the coming few decades, model organisms will continue to provide insights into
replication, transcription, … many other aspects of cell biology, biochemistry, and
physiology, because they offer the keenest methods of analysis”
o E.g. new disease gene in a conserved cellular process
 “Model organisms will increasingly be used for the direct investigation of medical
problems that seemingly have little to do with them.”
o E.g. neurodegeneration disorders (E.g. Alzheimer’s) involving protein
folding/aggregation
 “Model organisms will remain at the forefront for the forseeable future in efforts to sort
out biological complexity and achieve a more quantitative understanding of life
processes…”
o Thus particularly relevant for systems biology
 “Model organisms offer the best hope for coming to grips with the breadth of genetic
diversity and the depth of its consequences”
o Needed for tailored and personalized medicine
 “Model organisms will remain the proving ground for developing new technologies,
which typically spread quickly”
o E.g. yeast, two hybrid screens, genetic manipulation

2.4 Single-cell Organisms

2.4.1 E. coli
 There are many laboratory strains, a common strain is K12
o Non-pathogenic bacterium
 Fast doubling time and simple growth requirements, only ~4,000 genes which can be
manipulated with relative ease
 Arguably “the best understood species on earth”
o Allows us to ask very deep questions about life, and stand on a mountain of prior
research
 Understanding how molecular networks and circuits give rise to biological
function (e.g. Shen-Orr et al., 2002, Nat Genetics)

20
BE176 – Cellular and Molecular Biology Notes Module 2

 Understanding evolutionary processes (e.g. Barrick et al., 2009, Nature)

 Molecular motors such as flagellum and proton pumps
 Also used as a tool
o E.g. Production of DNA plasmids or proteins of interest in molecular biology
research
 Disadvantages
o Doesn’t have a nucleus/chromatin
o Not all protein production mechanisms such as glycosylation
2.4.2 S. cerevisiae
 Baker’s yeast or budding yeast – same used to brew beer
 Eukaryotic organisms
o Cells have nuclei and many other basic cellular processes that have human cells
have
o Examples
 Mitochondria, cell cycle, trafficking through golgi and endoplasmic
reticulum, meiosis
 Much of our understanding of the cell cycle comes from studies on S.
cerevisiae
 A Nobel prize in 2001 was won based on such work
 Easy to genetically manipulate through homologous recombination
 Grow quickly and easily

2.5 Multi-cellular Organisms

2.5.1 C. elegans
 One of the simplest organisms with a nervous system
o Nervous system is completely mapped
 Can be used to study reproduction and development
o Developmental fate of every single cell (1031 in adult males) is known
o 2002 Nobel Prize to Brenner, Horvitz and Sulston on organ development and
apoptosis
o 2006 Nobel Prize to Fire and Mello for RNA interference

21
BE176 – Cellular and Molecular Biology Notes Module 2

2.5.2 D. melanogaster
 Sexual reproduction and rapid generation time facilitates genetic studies
o Many human disease genes have homologs in drosophila
 Also has
o Vision
o More complex features and organs than C. elegans
2.5.3 D. rerio
 It is transparent during development and therefore amenable to study of development by
light microscopy
o Also used as a model for developmental toxicity
2.5.4 A. thaliana
 Like other model organisms is robust and has a short life cycle
 Used for genetics
o Has a small genome for plants
o Light sensing and circadian clocks
 Flowering

22
BE176 – Cellular and Molecular Biology Notes Module 2

2.6 Mammalian Systems

2.6.1 Cell Culture
 Cells from various tissue sources, most notably humans, can sometimes be cultured in a
dish or flask
o American Type Culture Collection (ATCC) has thousands of human cell lines
 It is much easier to study cells in culture than whole organisms
o Can do more perturbation and measurement techniques
o Quicker
o Cheaper
 Most cell lines are “transformed” meaning they grow indefinitely in cell culture, and
there are some artifacts due to that
o A large majority are derived from tumors
2.6.2 M. musculus
 Quickly reproducing and amenable to genetic manipulation
o Preferred genetic model for mammalian systems
o A variety of strains with gene knock out, knock-in and conditional expression
exist
 Strains exist without an immune system to facilitate studies involving human cell line
xenografts
o E.g. tumor growth and drug response
2.6.3 R. norvegicus
 Similar to mice
 Differences
o Not as amenable to genetic manipulation as mice
o Regarded as more intelligent than mice
o Preferred in neuroscience and behavioral experiments

2.7 Computational Models as a Biological Model System

 One of the main motivations for systems biology is to build computational models that
are good enough to be considered another type of model system

23
BE176 – Cellular and Molecular Biology Notes Module 2

o Not yet practical

o Most relevant effort: A Whole-Cell Computational Model Predicts Phenotype
from Genotype
 Potential applications
o Design microorganisms that
 Do a better job a remediation of waste water
 Produce a certain complement of small organic molecules from raw
materials
o Predict efficacy and toxicity of potential drugs prior to expensive clinical trials
 Get rid of potential problem drugs earlier in the development pipeline
o Identify patient populations that are more or less likely to respond to a drug
 “pharmacogenomics”
 Personalized medicine, e.g. in cancer
o Predict new uses for already approved drugs – repurposing
 New drug combinations to improve efficacy or reduce toxicity
 Using a cancer drug for arthritis – methotrexate

24
BE176 – Cellular and Molecular Biology Notes Module 2

Topic 3: Experimental Perturbations

3.1 Outline
3.1.1 “Basal” Perturbations
 Genetic
 Non-genetic

3.1.2 “Acute” Perturbations

 Compound-based (pharmacological and biological)
 Light-based

3.1.3 We focus on mammalian cell line and cell culture studies, but many of the techniques we
describe can be applied to other model systems (with reasonable alteration)
 There are lots of techniques in mouse genetics that will not be covered

3.2 Basal vs. Acute Perturbations

3.2.1 Basal Perturbation
 An alteration that is permanently present (or at least can be considered permanent relative
to the time scale of the experiment), and for which one does not measure dynamic
response
 Often followed by an acute perturbation
 Can also be thought of as “chronic”

3.2.2 Acute Perturbation

 An alteration to the biological system that induces rapid changes (relative to the time
scale of the experiment)
 One is often interested in a specific time point or an entire time course of many time
points post-perturbation

3.3 Basal Perturbations

3.3.1 Genetic – Mouse Embryonic Fibroblasts (MEFs) or Embryonic Stem (ES) Cells
 Wide variety of genetic knockouts are available

25
BE176 – Cellular and Molecular Biology Notes Module 2

 Many knockouts are embryonic lethal, but one can usually at least obtain fibroblasts or
ES cells from the embryo
o They can be grown in culture
 Those can be useful for exploring the molecular functions of particular gene products
 They can also be useful for negative and positive controls for other types of experiments
 ES cells can be differentiated into a variety of other cell types (growing field)
o Induced pluripotent stem (iPS) cells are similar but can be derived for adult
human cells (e.g. fibroblasts)

3.3.2 Genetic – Plasmid-Based Selection

 Plasmids can contain “resistance genes”
o Code for a protein that metabolizes a toxic drug
o Puromycin, G418 (similar to neomycin), blasticidin, zeocin, hygromycin B
 By transfecting the plasmid and treating cells with the drug, only cells that incorporated
the plasmid’s resistance gene will survive
o The plasmid gets randomly incorporated into the genome
o Linearizing the plasmid by making a single restriction enzyme cut improves
efficiency
 Incorporating the plasmid into a lentivirus can improve efficiency
 One can also use fluorescent proteins combined with flow sorting as an alternative
(although there is no active selection process)

3.3.3 Genetic – Genome Engineering

 Genome engineering – directed modification of particular loci
 General strategy – induce DNA double strand breaks and then rely on
o Non-homologous end joining for repair, which typically disrupts gene function
o Homologous recombination, which places a provided double stranded DNA
template where the break occurred
 Recombination enzymes
o Flp recombinase and CRE/Lox systems – these recombination enzymes
 TALENS and ZFNs

26
BE176 – Cellular and Molecular Biology Notes Module 2

o Requires protein engineering to achieve DNA sequence specificity of ~12-24 bp

and do not always cut in the same location
o Can only reasonably target one locus at a time
o Thus they are somewhat difficult to use and target
 Latest – CRISPR/Cas9 (Ran et al., Nat Protocols, 2013 and References therein)
o Uses a 20 bp “guide RNA” (gRNA) to bring the Cas9 nuclease to DNA which is
complementary to the guide RNA
 Allows simple and cheap targeting by oligo synthesis
 Can be targeted almost anywhere in the genome quite specifically (20 bp
can be reasonably unique)
 Tools at [Link]
o Wild type Cas9 vs. “nickase” Cas9
 Wild type catalyzes a full double strand break where the gRNA dictates
 Nickase Cas9 catalyzes a single strand break, therefore one needs two
“nicks” to make the double strand break

3.3.4 Non-Genetic – RNA interference

 Discovered in 1998 by Fire and Mello (Fire et al., Nature, 1998) – awarded Noble prize
in 2006
 A short (~20 bp) duplex RNA is directed to complementary transcripts, which are then
degraded
o Duplex can be small interfering RNA (siRNA) – two independent strands, or
short hairpin (shRNA) – one strand that hybridizes to itself with a stem loop at
one end (looks like a hairpin)
 This “knocks down” expression of the corresponding genes
o Similar pathways process micro RNAs (miRNA) that are produced endogenously
and can inhibit translation and degrade mRNAs
 Efficiency of knockdown widely varies across genes and cell types
 Need to use two independent sequences to control for off-target effects

27
BE176 – Cellular and Molecular Biology Notes Module 2

o Gold standard is a “rescue” experiment, where an RNAi resistant transcript is

ectopically expressed from a plasmid (resistant through engineered silent
mutations)
 Can be done in a transient or stable manner
 Can be inducible
3.3.5 Non-Genetic – Transcription Rate
 A variety of systems exist to control transcription rate with small molecules
o Too many to cover, but all work similarly to a certain extent
 One common system is with tetracycline (or more commonly its more stable and higher
affinity analog doxycycline)
 Tet-on system
o Cells expressing the tet repressor, and with the gene of interest downstream of the
tet repressor binding sites
o The tet-repressor only binds DNA when it is not bound to tetracycline
 There are also “Tet-off” systems

3.3.6 Non-Genetic – Protein Half-Life

 A few systems allow modulation of protein half-life with small molecules
 They all involve some kind of alteration to the protein-of-interest
 Examples
o DD domain/Shield1 (Banaszynski et al., Cell, 2006)
 Shield1 enters live cells, binds to the DD domain, and increases protein
stability
o auxin/AID (Holland et al., PNAS, 2012)
 Auxin (IAA) works similar to Shield1 but decreases protein stability
 However it requires ectopic expression of TIR1

3.4 Acute Perturbations

3.4.1 Pharmacological or Biological Compounds – Pipette-Based
 Pharmacological

28
BE176 – Cellular and Molecular Biology Notes Module 2

o A compound that is not naturally occurring in the system of interest but interacts
with the model system of interest
o E.g. a drug
 Biological
o A compound that normally interacts with the system of interest in a biological
context
o E.g. a growth factor
 Types of input
o Step input – Treatment with a compound at a certain constant concentration

o Pulse-Chase Input – Treatment with a compound at a certain concentration for a

specified time period, followed by a second concentration (usually zero)

3.4.2 Pharmacological or Biological Compounds – Pipette-Based

 Although it is not common or very simple, one can use pump perfusion or pump-based
systems to create complex time dependent inputs
 Microfluidic designs can give enormous flexibility here
 Types
o Ramp – Treatment concentration linearly ramps up or down with time

o Wave – treatment concentration oscillates in time with a fixed amplitude and

period

29
BE176 – Cellular and Molecular Biology Notes Module 2

3.4.3 Light-Based
 Optogenetics – using light-sensitive proteins to control biochemical activities
 First application was ion channels in neuroscience
 Has been extended to various light-sensitive protein-protein interactions and transcription

30
BE176 – Cellular and Molecular Biology Notes Module 2

Topic 4: Introduction to Gene Expression Analysis

4.1 Gene Expression
 Process by which information from a gene is used in the synthesis of a functional gene
product
o Included are types of RNA (not just limited to proteins)
 End products are proteins and non-coding RNA
 Genotype to phenotype
o Phenotype – observable traits
o Genotype – genetic makeup
4.1.1 Central Dogma
 DNA replication – DNA is doubled during cellular division (DNA to DNA)
 Transcription – DNA to RNA by RNA polymerase
o Production of an RNA copy from a DNA strand performed by RNA polymerases
o In bacteria, RNA polymerase binds to the Pribnow box (TATAAT) with the help
of the sigma factor
o For eukaryotes, RNA polymerase binds to the TATA box
 RNA pol. II transcribed protein-coding genes
 Requires transcription factors to bind to the promoter region 30 bp
downstream from the TATA box
o RNA pol. requires transcription factors and sigma factors
o Sigma factors bind to the promoter region
 Reverse Transcription – RNA to DNA by reverse transcriptase (never creates fully intact
DNA)
 RNA replication – RNA to RNA to create (-) sense RNA from (+) sense RNA
 Translation – mRNA to proteins
 mRNA processing
o eukaryotic processing of mRNA
o Transforms Pre-mRNA to mRNA

31
BE176 – Cellular and Molecular Biology Notes Module 2

o splicing by spliceosomes removes and releases introns as lariat structures by two

transesterification reactions
o 5’ cap – Addition of 7-methylG then bound by cap binding complex heterodimer
to aid export mRNA from the nuclear lamina
o Poly-A tail – 3’ cleavage and polyadenylation; occur if polyadenylation signal
sequence (5’-AAUAAA-3’) is present; poly(A) tail is bound by multiple poly(A)-
binding proteins (PABPs) necessary for mRNA export
 Alternative splicing
o Differences in splicing can change the resultant protein by exon skipping
o Variants of the same protein are referred to as isoforms
o Regulation of alternative splicing occurs at the transcription level
 Methylation sites also regulate which mRNA are formed

4.2 Regulation of gene expression

 Gene expression may be regulated at the following levels
o Protein
o mRNA
o DNA
 Control the functional gene product in two fronts
o Amount
o Timing
 Example: Progeria
o Disease that causes rapid aging
o Mutation of (LmNA) gene messes with the timing of gene expression, causing
rapid aging
 Example: tortoise shell or calico cats
o Majority are female
o Genes for coat colorization in cats are located in the X chromosome
 X inactivation occurs randomly, causing a mixture of coat coloration

32
BE176 – Cellular and Molecular Biology Notes Module 2

 Male cats are usually whole color coated because X inactivation does not
give rise to patches of coloration
o Mutation is due to X-inactivation
 One X chromosome is inactivated randomly, leaving only a single X
chromosome active
 Inactivated X chromosomes clump together in a concentrated area in the
nucleus called Barr bodies

4.2.1 General types of genes

 Constitutive gene – gene that is transcribed continually as opposed to a facultative gene
 Housekeeping gene – gene that is required to maintain basic cellular function; is typically
expressed in all cell types of an organism
 Facultative gene – gene that is transcribed discontinuously only when needed; cells
receive signals from their surroundings to begin transcription
 Inducible gene – can be activated by the presence of signal molecules
 Repressible genes- can be inactivated by signal molecules

4.2.2 Measurement of Gene Expression

 Some Applications
o Identify viral infection of a cell (viral protein expression)
o Determine an individual’s susceptibility to cancer (oncogene expression)
o Find if a bacterium is resistant to penicillin (beta-lactamase expression)
4.2.3 Population Average vs. Single Cell Techniques
 Population average techniques require a large amount of starting material
o Their output reflects the average over typically ~1 million cells or more
 Singe cell techniques are sensitive enough to detect analytes from individual cells
o A main challenge is separating measurement noise from true cell-to-cell
variability
o Becoming increasingly appreciated that many important phenomena are
uncovered by observing at the single cell level

33
BE176 – Cellular and Molecular Biology Notes Module 2

4.3 Studying role of genes in the development and diseases at the transcription
level

 Most sensitive relative to the techniques

 Discussed are the most common methods to study role of genes at the RNA level
4.3.1 Timeline
 1970s – Northern Blot
 1980s – qPCR
 1990s – Microarray
 Present – RNA-Seq

4.4 Northern Blot

 Low throughput technique
 Focuses on the detection of RNA sequences
o Abundance of RNA sequences can be used to measure the level of gene
expression
 Detect if certain genes are transcribed or not
 Can only be used for semi-quantitative measures of gene expression
o Based on the opacity of bands detected
o Not completely quantitative
 Southern Blot – only detects which DNA genes are present

4.5 PCR
 A technique that amplifies certain regions of interest in the DNA
 Does not amplify the entirety of the DNA
 Primers are utilized to allow for polymerase (TAQ pol.) to amplify a specific part of the
DNA
 In vitro DNA replication
 Automatically cycles temperature to allow for denaturation, annealing, and extension
 Can be used to identify the region of interest in the DNA molecule
 Amplifies sections of DNA for further testing

34
BE176 – Cellular and Molecular Biology Notes Module 2

4.5.1 Types of PCR

 Conventional PCR endpoint analysis
o Detects the presence of a sequence of interest (qualitative, or at most semi-
quantitative)
o Results can only be analyzed after gel electrophoresis
o Example: Detection of STRs in genetic testing, pathological deletion mutation
o STR (Short Tandem Repeats) Analysis
 The TH01 is an example of STR
 the locus of which contains repeats of TCAT
 CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA
 This example has 6 TCAT repeats
 There are more than 20 known TH01 alleles
 Each individual inherits 1 allele from each parent
 STR regions can be amplified using PCR and run through gel
electrophoresis
o VNTR (Variable Number of Tandem Repeats)
 Has larger sequence of repeats relative to STR (20-60 bps)
o RFLP (Restriction Fragment Length Polymorphism)
 Used to identify DNA fragments based on unique patterns of restriction
enzyme cutting in specific regions of DNA and see them in gel
o SNPs (Single Nucleotide Polymorphism)
 Single nucleotide changes in genome
 SNPs occurs once in 1% of the population
o Example Application: Crime Scene investigation
 Detects multiple loci
 The Power of Discrimination with the number of loci profiled
 Combined DNA Index System (CODIS) – federally maintained database
used by law enforcement officials
o GMO Detection
 Looks for promoters CaMV 35S and terminator nopaline synthase (NOS)
terminator Agrobacterium tumerfaciens
35
BE176 – Cellular and Molecular Biology Notes Module 2

 PSII chloroplast gene is used to check if DNA extraction is successful

 Multiplex PCR – Amplifies more than a single gene
 qPCR (Quantitative PCR or Real Time PCR)
o Allows the accumulation of amplicon to be detected and measured in real time as
the reaction progresses
 Conventional PCR – Amplicon is detected only by endpoint analysis using
agarose gel
o Advantage: allows the determination of starting template copy number
o Generates an Amplification plot

o At cycle 10, the fluorescence is difficult to detect

o At higher cycle numbers, fluorescence intensity rapidly increases
o CT – Threshold cycle number
 Number of cycles where fluorescence can be detected
 Low CT = High concentration
 Linear standard curve for CT and starting concentration indicates good
efficiency
o Legend
 Red is high concentration and Yellow is low conc.
 Grey indicates no detected amplicon

36
BE176 – Cellular and Molecular Biology Notes Module 2

o Hallmarks of an optimized qPCR assay

 Linear standard curve (R2 > 0.98)
 High amplification efficiency (90-105%)
 Consistency across replicate reactions
o Efficiency formula
 E = 10-1/slope – 1
 An efficiency of 100% indicates that the template doubles after ach
thermal cycle during exponential amplification
 Experimental factors such as the length, secondary structure, inhibitors,
and GC content of the amplicon can influence efficiency
o Fluorescence detection systems
 SYBR Green – as amplification occurs, the fluorescence signal increases;
SYBR binds to the double-stranded amplicons nonspecifically
 TaqMan – employs a TaqMan probe downstream from the point of
attachment; TaqMan probe is made of reporter dye and quencher; Once
elongation is initiated, the probe is cleaved and releases the reporter dye,
creating fluorescence
o Other detection chemistries
 Mol. Beacons
 Hybridization probes
 Eclipse probes
 Amplifluor
o Primer design considerations
 Design primers are 18-28 nucleotides in length (in general)
 Avoid stretches of repeated nucleotides
 Aim for 50% GC content, which helps to prevent mismatch stabilization
 choose primers that have compatible Tm values (within 1 deg. C of each
other) a
 avoid sequence complementarity between primers
o Absolute Quantification

37
BE176 – Cellular and Molecular Biology Notes Module 2

 Real -time PCR analysis of choice for researchers who need to determine
the actual copy number of target investigation
o Relative Quantification
 Answers the question “what is the fold difference”
 Provides data on the changes or differences in gene expression
 Compares the levels of gene expression
 Example applications: developmental biology, disease research
o Normalization Methods
 Against unit mass
 To a reference gene
 Examples: GADPH, beta-actin
 Method of normalization against unit mass
 Ratio(test calibrator) = ECT(calibrator) - CT(Test)
 Sample Problem

38
BE176 – Cellular and Molecular Biology Notes Module 2

 2-Δ ΔCT or Livak Method

39
BE176 – Cellular and Molecular Biology Notes Module 2

 ΔCT Method
 Normalized against reference gene
 Pfaffl Method
 Used when amplification efficiencies are not similar
Δ C T ,target (calibrator−test)
(E target )
Ratio= ,ref (calibrator −test)
( Eref )Δ C T

40
BE176 – Cellular and Molecular Biology Notes Module 2

 Other types of PCR

o Multiplex PCR
o Long-range PCR
o Single-cell PCR
o Methylation-specific PCR (MSP)
o RACE: Rapid Amplification of cDNA Ends
o In situ PCR

4.6 Microarray
 Predominantly used for measuring transcript levels
 Each spot has a different oligonucleotide
 Complementary oligos bind to each spot by base pairing, which is observable as
increased fluorescence
 There are also bead-bead methods (as opposed to slides or chips – illumina)
 Workhorse in the 2000s, but next generation sequencing is emerging as superior
(although still more expensive but probably not for long)

41