Mtap2 W21 Molbio

MOLECULAR BIOLOGY W E E K｜21
MODULE WRITER: JEFFERYL KAE PANDAC MTAP 2
OUTLINE ● Institutional and personal security measures designed to

I. Biosafety and Waste Management prevent the loss, theft, misuse, diversion, or intentional
II. Review of DNA and RNA release of pathogens and toxins
A. Structure and Function
B. Central Dogma of Molecular Biology
C. Replication, Transcription, Translation
III. DNA and RNA Isolation Techniques
IV. DNA Hybridization Techniques
V. Polymerase Chain Reaction
A. Principle
B. PCR Reagents BIOSAFETY
VI. DNA Sequencing ● Accidental/Unintentional release of biological agents
VII. Proteins
● Protecting people from bad bugs.
A. Structure and Function
B. Isolation Techniques
C. Techniques in Protein Analysis BIOSECURITY
● Intentional/Deliberate release of biological agents
● Protecting bad bugs from bad people.
BIOSAFETY AND WASTE MANAGEMENT
● Possible to generate waste with different classifications “BIOSAFETY and BIOSECURITY differs in their INTENT”
Biological Agent Biosafety vs. Biosecurity Issue

● Any microbial entity, cellular or non-cellular, naturally 1. Recapping of needles - Biosafety
occurring or engineered, capable of replication or of ● Needles may contain biological agent, and you
transferring genetic material that may be able to do not have the intent to spread the biological
provoke infection, allergy, toxicity, or other adverse agent
effects in humans, animals, or plants 2. Your lab technologist is a member of a terrorist group -
● Examples: bacteria, fungi, viruses, viroids, Biosecurity
endo/ectoparasites ○ The member may deliberately use the
biological agents.
Biological Material 3. Lab personnel not provided with PPEs - Biosafety
● Any material comprised of, containing, or that may ○ When lab managers fail to provide necessary
contain biological agents and/or their harmful products, PPEs, the chances for the person to acquire lab
such as toxins and allergens infections is present
○ Can be PPE, different materials in the lab, or 4. Wastes from the laboratory are not segregated -
culture plates Biosafety
○ The potential spread of biological agent is
there, and the intent is unintentional
5. Staff with a huge debt - Biosafety and Biosecurity
○ Depends on how you see it, it is biosafety when
the person have a huge debt, he may be
always agitated, worried or cannot concentrate
Biohazard and potentially acquire the agent; but can be an
● Potential source of harm caused by biological issue in biosecurity when the person contact
materials people from the terrorist group and sell the
○ Can be an event or activity that may potentially agent.
source of harm or any area in the lab that can
be source of biological material which contain Laboratory Biosafety
biological agents ● PROTECT USERS OF THE LABORATORY
● PROTECT THOSE OUTSIDE THE LABORATORY
Laboratory Biosafety ● PROTECT THE ENVIRONMENT
● Containment principles, technologies, and practices
implemented to prevent unintentional/accidental Principles of Biosafety
exposure to pathogens and toxins, or their 1. Practices and procedures
unintentional/accidental release 2. Standard practices
○ Most important concept
Laboratory Biosecurity
bacani.miranda.payumo.pineda.policarpio.punsalan.rubiano.santos 1
■ Even if you have the correct but if you
BSL2 & BSL3
do not practice standard procedures
(used for
in the lab, it is useless. COVID-19)
○ Aware of potential hazard
○ Trained and proficient in techniques Maximum Dangerous As Level 3 plus Class III BSC,
3. Special practices and considerations Containment – pathogen units airlock entry, or positive
○ Safety equipment BSL4 shower exit, pressure suits
○ Facility design and construction special waste in conjunction
Used by the disposal with class II
○ Increasing levels of protection CDC BSCs,
double-ended
1. Safety equipment Highly infectious autoclave
● Primary containment barrier to minimize biologic agents (through the
example: Ebola, wall), filtered
exposure to hazard
smallpox air
○ Stand between you and the biological
agent
● Engineering controls/equipment Biological Waste or Biohazardous Waste
● Personal Protective Equipment (PPE) ● a.k.a. infectious waste or biomedical waste
● Any material that contains or has been contaminated by
● Example: Covered or ventilated animal cage
a biohazardous agent
systems ● Any waste containing infectious materials or potentially
infectious substances such as blood
2. Facility Design and Construction ○ Blood may contain transmissible infections
● Secondary barrier/engineering controls such as HIV/AIDS, Hepatitis B
● Contributes to worker protection
● Protects those who are outside the laboratory Cultures and Stocks of Infectious Agents
● Specimen from medical and biological laboratories
(environment and neighborhood) ● Cultures and stocks from infectious agents from clinical,
● Examples: building and laboratory design, research, and industrial laboratories
ventilation, autoclaves, cage wash facilities ● Before discarding, they must be decontaminated
3. Increasing Levels of Protection Human Blood and Blood Products

● Biosafety Levels 1-4 ● Wastes containing recognizable fluid blood or blood
products (plasma, platelets, cryoprecipitates, etc.),
○ Increasing levels of employee and
containers with blood, blood from animals
environmental protection
○ Guideline for working safely in Pathological Wastes
research and clinical laboratory ● Human surgery specimen or tissues, including those
facilities fixed in fixatives
● Will depend on the organisms being handled ● Animal parts, tissues, fluids, or carcasses
○ If they will potentially cause harm to
Contaminated Sharps
your people?
● Contaminated hypodermic needles, syringes, scalpel
○ What PPE is suitable for them? blades, Pasteur pipettes, and broken glass items (vials,
tubes, etc.)
Biosafety Levels ○ Should be stored in a leak-proof/
● Combination of laboratory practices and procedures, puncture-proof container
safety equipment (primary barriers), and laboratory ○ Micropipette tips (blue, yellow, white) - recently
facilities (secondary barriers) added as they may puncture mats
● Also referred to as Containment Levels Other Biological Wastes
● Isolation wastes: generated by hospitalized patients
who are isolated to protect others from communicable
BIOSAFETY LABORATORY LABORATORY SAFETY diseases
LEVEL TYPE PRACTICES EQUIPMENT
● Pharmaceutical wastes: expired, unused,
split/contaminated pharm products, drugs, vaccines, and
Basic – BSL1 Basic teaching, GMT None; open sera
research bench work
Mixed Waste
Basic – BSL2 Primary health GMT plus Open bench ● Any hazardous waste which contains more than one
services; protective plus BSC for principal class of hazardous waste
diagnostic clothing, potential ○ Chemical, radioactive, biological - when you
services; biohazard sign aerosols have combination of these waste, it is called as
research Mixed waste
● If different types of waste are mixed, the waste mixture is
Containment – Special Level 2 plus BSC and/or treated according to the Mixed Waste Hierarchy
BSL3 diagnostic special clothing primary ○ Highest: radioactive waste > chemical >
services, controlled devices for all biological
research access, activities
directional
airflow
● Segregation is the key to any effective waste
management
● Without effective segregation system, the waste must be
considered as hazardous
○ Domestic waste may be already mixed to
hazardous waste
Color Coding System of Waste Segregation

● Black: Non-infectious, dry general waste
● Green: Non-infectious, wet waste
● Yellow: Infectious waste
● Yellow Bag w/ Black Band: Chemical & Pharmaceutical
Waste
● Orange: Radioactive waste kept in leaded container
○ Lead - prevent escape of radioactive waste
Rule of thumb in dealing with segregation errors

● If general and hazardous wastes are accidentally
mixed, the mixture should be treated as hazardous
waste
INTERPRETATION: ○ To also protect those people working in the
● Whenever there is a radioactive waste present, treat the transport facility
waste as radioactive
● Chemical + Biological + Radioactive = radioactive Packaging
waste ● Select packing materials that are appropriate for the type
● Biological + Radioactive = radioactive waste of waste
● Chemical + Radioactive = radioactive waste ○ Plastic bags for solid or semi-solid infectious
● Chemical + Biological waste = chemical waste waste
○ Bottles, flasks, or tanks for liquid and
Local and National Regulations on Waste Management semi-liquid wastes
○ Puncture- and leak-proof plastics for sharps
■ Do not throw sharps in plastic bags,
AGENCY REGULATION NO. FULL TITLE as they can be punctured easily
● Label Properly
DOH RA 4426 Hospital Licensure Act
Handling and Storage of Biological Wastes
DOH PD 856 The Code on Sanitation ● Collection of infectious waste must be done by trained
of the Philippines utility personnel
○ To limit the spread of biological agent
● Protective measures for the personnel handling
DOH DC 156-C series Guidelines on Hospital infectious wastes
of 1993 Waste management ○ Immunization (tetanus, Hepatitis B)
○ PPE (mask, heavy-duty gloves, protective
DENR PD 1586 Environment Impact clothing, safety boots)
Statement System ● Collection must be done on a regular basis
○ Must not be stored for a long time as this may
DENR RA 8749 Clean Air Act of 1999 encourage the growth of bacteria, fungi, and
other microorganisms which then ultimately
result in foul-smelling waste.
DENR RA 9003 Ecological Solid Waste
Management Act of 2000 Storage
● Storage temperature and duration are important
DENR PD 984 Pollution Control Law considerations
○ Must be well-ventilated
DENR RA 6969 An Act To Control Toxic ● Warmer temperatures cause higher rates of microbial
Substances and growth and putrefaction, resulting in odor problems
Hazardous Nuclear ● Consider volume of waste generated in the allocation or
Waste designation of storage area
○ Prepare a facility that can accomodate the
volume of waste generated.
Waste Management Process
● Segregation Transport
● Packaging ● Frequent disinfection of carts used to transfer wastes
● Handling & Storage within the facility
● Transportation ● Placement of all infectious wastes into rigid or semi-rigid
● Treatment & Disposal containers before offsite transport
● Use of closed, leak-proof trucks or dumpsters
Segregation ● Use of appropriate hazard symbols
● Separation of different classifications of wastes from ○ Important for the safety of those handling the
each other, beginning at the point of generation, and waste
maintaining the separation during storage and transport, ● Properly document transport of biological waste for
until treatment traceability purposes
○ Most important step
Treatment & Disposal ○ They contain letter “y”
● On-site treatment of biological waste include:
○ Autoclaving
■ For culture plates and other materials
that may potentially contain
organisms.
○ Chemical disinfection (10% hypochlorite
overnight before drain disposal)
○ Microwave irradiation (effective when there is
moisture in the waste)
○ Incineration
Other Waste Treatments

● Biological process – use of enzyme mixture to
decontaminate waste to render the agent less dangerous
● Radiation process – breaking the DNA molecules of
contaminating organisms
● Encapsulation – mixing waste with immobilizing
material (plastic foam, cement mortar); useful for sharps DNA Base Pairs
and pharmaceuticals ● Adenine with Thymine (A with T)
○ “Apple in the Tree”
● Guanine with Cytosine (G with C)
Review of DNA and RNA ○ “Car in the Garage”
● Note: each base is attached to a sugar molecule and a
phosphate molecule
Friedrich Miescher
● First isolated DNA in 1869 DNA Double Helix
when he discovered ● Nucleotides are arranged in 2 long strands that form a
microscopic substances spiral called a double helix
○ Most are found in the nucleus
from pus among surgical ● Held together by hydrogen bonds between the
bandages complementary nitrogenous bases on either side of the
● DNA was first termed as strand
nuclein
Ribonucleic Acid (RNA)
● Transfers the genetic code from the nucleus to the
Francis Crick & James Watson ribosomes during protein synthesis
● Introduced the currently ○ DNA molecules found in the nucleus cannot
accepted model of the synthesize protein directly → must be
DNA (double helix) in converted into smaller molecules (RNA) to go
1953 out the nucleus and be transferred to
ribosomes.
● Awarded a price in
● Important roles in protein synthesis, communicating
biochemistry for the cellular signals, and catalyzing some chemical reactions
discovery.
RNA Structure
● Single-stranded
Deoxyribonucleic acid (DNA) ○ Making it less stable
● Hereditary material in humans and almost all other than the DNA
organisms molecule, can easily
○ Some viruses may only contain RNA (not all be degraded.
organisms has DNA) ○ Observe strict
● Most are found in the nucleus (nuclear DNA); a small protocols when
amount can be found in the mitochondria (mitochondrial handling RNA due to
DNA) its unstable
● Made up of four types of nucleotides characteristic
● Formed by a pentose sugar
Nucleotides: Building Blocks of DNA ribose backbone
- Makes up the DNA ● Nucleobases:
- Has basic structure: has a sugar-phosphate backbone, ○ Adenine
nitrogenous bases (purines and pyrimidines), and base ○ Guanine
pairs ○ Cytosine
○ Uracil
Nitrogenous Bases
● Purine – Adenine (A) and Guanine (G)
● Pyrimidine – Cytosine (C) and Thymine (T)
DNA RNA
Function Carries genetic Converts genetic

information. information from DNA
Serves as template into proteins. Some
for synthesis of RNA have regulatory or
structural functions.
Source of genetic
information in RNA
viruses.
Location Nucleus (except Nucleus and cytoplasm

mitochondrial DNA)
Composition Repeating Repeating nucleotides

Messenger RNA (mRNA) nucleotides linked by linked by
● Responsible for carrying information about a protein phosphodiester phosphodiester bonds
sequence to the ribosomes bonds between 5” between 5” phosphate
● Product of transcription from the DNA molecule inside phosphate group of 1 group of 1 sugar and 3’
the nucleus. sugar and 3’ hydroxyl hydroxyl group of next.
group of next.
Transfer RNA (tRNA)
● Small RNA with the size of about 80 nucleotides
Sugar Deoxyribose Ribose
● Transfers amino acids to growing polypeptide chains at
the ribosomal site of protein synthesis
○ Can only be find in the ribosomes Pyrimidines C, T C, U
● Important regions:
○ Anticodon Purines A, G A, G
○ Site for amino acid attachment
Ribosomal RNA (rRNA)

● Comprise majority of the RNA found among typical Replication, Transcription, Translation
eukaryotic cells
● Catalytic component of the ribosome DNA & RNA ISOLATION TECHNIQUES
● rRNA and proteins combine to form the ribosome
ISOLATION OF DNA
Structure and Function Preparing the Sample
Central Dogma of Molecular Biology ● Sources of nucleic acid (NA) samples: human,
● Describes the flow of genetic information from DNA to fungal, bacterial, viral sources
RNA to protein ● Initial steps of NA isolation depend on the
● The DNA is found in the nucleus and some mitochondria. nature of starting material and the test method
● DNA will undergo replication, inside the nucleus, it has ● Collection of DNA:
first to be converted into smaller molecules (mRNA)
○ Blood (higher yield)
known as transcription (DNA → mRNA). mRNA takes
instructions from the DNA molecule. ○ Nail clippings
○ Transcription happens inside the nucleus. ○ Swab of epithelial cells
● mRNA leaves the nucleus and goes to the cytoplasm,
specifically, in the ribosome so that the information in
mRNA can be converted into proteins, also known as STOPPER CONTENT USE
translation. COLOR
○ Translation happens in the ribosomes.
● Some organisms such as viruses that contain only RNA, Lavender EDTA Isolation of DNA & detection of
their genetic material must be converted into DNA in the viruses.
PCR to be detected. 1 of preferred AC for blood
○ RNA to DNA is called reverse transcription. and bone marrow
White K2EDTA & gel Isolation of plasma. Gel forms

barrier barrier between plasma & cells
Blue/Black Sodium Isolation of mononuclear cells.

citrate, gel, Gel forms barrier between
density mononuclear cells (monocytes
gradient fluid and lymphocytes) in plasma &
RBCs/ granulocytes
Yellow Acid Citrate Enhanced WBC recovery for

Dextrose several days after collection.
(ACD) 1 of preferred AC for blood
COMPARISON BETWEEN DNA AND RNA and bone marrow
○ Must be concentrated into solid-phase
Green Heparin Generally not recommended concentration techniques to acquire optimum
as it inhibits polymerase;
amount of DNA
unacceptable for testing that
involves PCR ● Solid tumors and transplanted organs release cells,
exosomes, and NA into the bloodstream
● Before isolation, concentration of NA is required through
Sources of Nucleic Acid (NA) solid-phase collection techniques
1. Bacteria and Fungi
● Cell walls must be broken down because the goal is to 4. Tissue Samples
isolate DNA and the DNA is inside the organism ● Fresh, frozen tissues need to be dissociated first before
surrounded by a thick cell wall. DNA isolation
● Cell walls are thick and must be broken down using the ○ Dissociation - perform mechanical grinding/
following methods: macerate the tissue.
○ Enzymatic treatment ● Fixed, embedded tissues should be deparaffinized first
○ Mechanical grinding with glass beads in xylene, then rehydrated in decreasing concentrations
○ Chemical treatment (detergent and strong of ethanol
base, EDTA, glucose) ● <100 bp or less can be obtained from fixed tissue
○ Commercial reagent kits (fast and simple) ● Extended proteinase K digestion may yield longer
■ Widely utilized, gives higher DNA DNA fragments
yield ○ Remedy
2. Nucleated Cells in Suspension (Blood and Bone Marrow Fixatives Influencing NA Quality
Aspirate) ● Buffered neutral formalin is the least DNA damaging
● NA comes mostly from WBCs ● Best: 10% NBF and acetone (has good relative quality of
● Differential density-gradient centrifugation nucleic acid)
○ Whole blood or bone marrow in saline is ● Mercury fixatives (Bouin’s and B5) are the worst for DNA
overlaid with Ficoll (highly branched sucrose recovery
polymer) which will act as separator based on ○ The DNA yeild can only be less than 0.1 kb
the differences in the density of different
components such as plasma, lymphocytes,
monocytes, granulocytes, and RBCs.
■ Separate RBCs and granulocytes
from other elements
○ Mononuclear WBCs are found below the
plasma, and above the Ficoll layer
○ Layer with mononuclear WBCs is removed and
washed with saline before NA isolation to
remove any contamination with RBCs and other
unnecessary cells.
DNA ISOLATION CHEMISTRIES
1. Organic Isolation Methods

● Accomplished using a combination of high salt, low pH,
and an organic mixture of phenol or chloroform
(dissolve hydrophobic contaminants from your tissue)
● Differential osmotic fragility of RBCs and WBCs ● CTAB (cetrimethylammonium bromide) separates
○ Whole blood or bone marrow is incubated with DNA from polysaccharide contamination (e.g., fungal
hypotonic saline to lyse RBCs sample)
○ WBCs are pelleted by centrifugation ○ Why fungat samples? Because they have thick
polysaccharide layers
● RNase is added to avoid RNA contamination
○ An enzyme will degrade RNA molecule
General Scheme of Organic DNA Isolation

● Generally used when the sample is fatty/hydrophobic.
3. Plasma
● Can be used to check for any genetic markers or
substances that will point to a possible solid tumor
● Used in liquid biopsy (precludes surgical biopsy)
Process: Process:
● Cells are lysed with NaOH, SDS ● Cell lysate is added into a column in high-salt buffer
○ Always start with lysis to release nucleic acids ● DNA adsorbs to the solid matrix in low pH
from the cells. ● Immobilized DNA is washed by buffer
○ Sodium Dodecyl Sulfonate (SDS): detergent ○ Usually washed twice (wash buffer 1 and wash
used in disrupting/lysing the cells buffer 2)
● Lysate is acidified with acetic acid and salts ○ Then remove the DNA molecules that have
○ Acidification used to remove cell debris adsorbed to the solid-phase matrix by the
● Hydrophobic contaminants is dissolved in phenol and process called elution
chloroform; DNA is in aqueous solution ○ Change the condition in the solid-phase matrix,
● DNA is precipitated by ethanol such that the DNA molecules will be
● The goal purpose here is to usually use organic isolation successfully elute or removed from the
methods to isolate DNA if your sample is fatty or solid-phase matrix. Then collect it to the last
hydrophobic in nature. tube.
○ The hydrophobicity will be removed by the ○ It is possible for the DNA molecules to
organic substances. adsorbed to the solid-phase matrix in the
presence of high-salt concentrations, it is also
2. Inorganic Isolation Methods possible to remove DNA molecules absorbing
● a.k.a. salting out to the solid-phase matrix in the elution step by
● Does not include the organic extraction step using a low-salt buffer
○ Samples being used are not fatty or ● DNA is eluted with low-salt buffer
non-hydrophobic in nature.
● Makes use of low pH and high salt conditions to Proteolytic Lysis of Fixed Material
precipitate proteins ● Cells or tissue may be washed by suspension and
● DNA can be precipitated by ethanol or isopropanolol, centrifugation in saline or isotonic buffers
pelleted and resuspended in TE buffer or water ● For simple screens, cells are lysed by detergents (SDS,
Triton)
General Scheme of Inorganic DNA Isolation ○ Sodium Dodecyl Sulfonate (SDS)
● For use in PCR amplification, cells are lysed by a mixture
of Tris buffer and proteinase K
○ digests protein and inactivates other enzymes
Process:
● Cells are lysed with Tris base, EDTA, and SDS
● Proteins are precipitated with sodium acetate
○ Proteins need to be precipitated because they
are considered as contaminants
● DNA is precipitated by isopropanolol or ethanol
3. Solid-Phase Isolation
● Use of solid matrices
○ e.g., silica-based products, glass beads,
columns
○ To bind and hold DNA for washing
○ Washing step - unique in this step, and absent
to other mentioned techniques. Ultimately give
a clearer DNA.
● Silica-based products effectively bind DNA in high-salt
conditions.
● Methodology employed for most robotic DNA isolation
systems Process:
● Follows a series of lysis, adsorption, washing, and ● Microdissect or remove the tissue that you want
elution steps ● Deparaffinize in xylene and then rehydrate the tissue
● Get the tissue and place it in the tube then do protenase
Isolation of DNA on Solid Media K diggestion
Rapid Extraction Methods
● Method most used in forensic application, as well as ● Transfer RNA (tRNA)
DNA isolation from samples spotted on storage cards ● Small nuclear RNA
○ e.g. of storage cards - Newborn screening filter
paper card
● Uses Chelex: cation-chelating resin RNA ISOLATION CHEMISTRIES
Storage of Extracted DNA: 1. Organic Isolation of RNA
● The lower the temperature, the longer the DNA will be a. Cell Lysis
viable. ● Performed in detergent/phenol in the presence
● Room Temperature – several months of high salt
● Refrigerated Temperature (2-8ºC or 1-6ºC) – 1 year ● RNase inhibitors, such as guanidium
● Frozen isothiocyanate or GITC, can be used instead
○ -20 degrees Celsius – 1 year of high-salt buffers
○ -70 degrees Celsius – 10 years b. Protein Extraction
● Main goal: Isolate or extract pure substance,
ISOLATION OF RNA either DNA or RNA
● RNA - single stranded; unstable ● Acid phenol: chloroform: isoamyl alcohol
● When working with RNA, strict precautions are (25:24:1)
observed to avoid RNA degradation. ● Chloroform denatures proteins and promotes
○ Slight changes in temperature may degrade phase separation.
RNA ● Isoamyl alcohol prevents foaming of lysate.
● Ubiquitous RNases degrade RNA and are hard to c. RNA Precipitation
eliminate ● Addition of ethanol or isopropanol
○ they tend to renature after autoclaving, and are ● RNA is resuspended in buffer or water
active at a wide temperature range
○ Ubiquitous - present everywhere, they are
present in palms, table tops
Guidelines to Observe when Handling RNA

● Allocate a separate RNase-free (RNF) area of the
laboratory intended for handling RNA
○ It is not allowed to handle RNA and DNA in the
same area.
■ This is because when you are
handling DNA, you will use RNAse
which is not suitable for RNA. 2. Solid-Phase Isolation of RNA
● Gloves must always be worn in the RNF area a. Cell Lysis
● Never touch disposables or consumables with ungloved ● Performed in detergent or phenol in the
hands presence of high salt
● Glassware must be cleaned thoroughly, and baked for ● RNase inhibitors, such as guanidium
4-6 hours at 400 degrees Celsius to inactivate RNases isothiocyanate or GITC, can be used instead of
○ Physical inactivation high-salt buffers
● Diethyl pyrocarbonate (DEPC) may be used to rinse b. Protein Extraction
glassware to inactivate RNases ● Acid phenol: chloroform: isoamyl alcohol
○ Chemical inactivation (25:24:1)
● Stabilize RNA from further metabolism or degradation ● Chloroform denatures proteins and promotes
after specimen collection phase separation
○ Snap-freezing sample in liquid nitrogen ● Isoamyl alcohol prevents foaming
■ Most commonly practiced technique c. RNA Precipitation
in molecular biology laboratory ● Addition of ethanol or isopropanol
○ Use of specialized collection tubes that ● RNA is resuspended in buffer or water
preserve RNA
○ Use of preservative reagents
■ Example: RNAlaterTM Stabilizing
Solution, ThermoFisher Scientific
Total RNA Composition Process:

● Ribosomal RNA (rRNA) ● Lyse the cells using detergent (SDS, Triton or other
○ Most abundant (80-90%) lysing buffers)
○ Two components: large and small rRNA ● Once you have your lysate, add it to a solid-phase matrix
● Messenger RNA (mRNA) (the environment must be low pH - acidic)
○ Second most abundant (2.5-5%) ● Once the RNA has already adsorb, then the washing
○ Detected as a faint background underlying step can proceed
the rRNA detected by AGE ● Wash (you can do 2 sets of washing)
● Elute the RNA by changing the condition inside the
solid-phase matrix nucleic acid
● Then precipitate the RNA using ethanol concentration.
Compare to
Proteolytic Lysis of Fixed Material standard
● RNA quality extracted from fixed, paraffin-embedded
tissues depend on the fixation process and pre-fixation Purity Spectrophotometry Nucleic acids DNA A260/A280
handling Absorbance absorb light at rate:
● Commercial reagents provide lysis and incubation @ 260 nm or 260 nm.
conditions that reverse formaldehyde modification of @ 280 nm 1.6 - 2.0 = good
RNA, and release RNA from tissue sections Absorption quality
peak for <1.6 = protein
protein is 280 contamination,
nm. specimen must
be reprocessed
>2 = possible
contamination
with RNA
RNA A260/A280
ratio:
>2= good quality
Gel electrophoresis Position of DNA:

or densitometry bands is High MW
related to MW. fragments = good
Lower MW quality
fragments
migrate RNA 28S/18S
Isolation of polyA (Messenger) RNA farthest from ratio:
● mRNA accounts for about 2.5-5% of the total RNA yield the point of
● mRNA extraction protocols employ single-stranded application. 2 = good quality
oligomers of thymine or uracil immobilized on a solid <2 = smaller
matrix (resin column, beads) fragments due to
● polyT or polyU oligomers will bind to the polyA tail found RNAse
on mRNA degradation
○ polyA tail - protect RNA from degradation.
○ Solid phase matrix must have single a single (check working
stranded polymer of thymine or uracil which area for possible
can bind to adenine contamination of
RNAse)
Nucleic Acid Hybridization
● polyA RNA is eluted using warmed, low-salt buffer NUCLEIC ACID HYBRIDIZATION
containing detergent
● the buffer will break the hydrogen bonds between the ● interaction between two single-stranded nucleic acid
mRNA and the column molecules to form a duplex (double-stranded) molecule
● Based on the complementary base pairing of their
Storage of Extracted RNA: respective sequences
● Stored suspended in ethanol for several months at -20 ● Base pairing may occur between:
degrees Celsius or long term at -70 degrees Celsius ○ complementary strands of DNA (DNA-DNA)
(RNA is less stable than DNA) ○ DNA and RNA (DNA-RNA)
○ Not advisable to be kept in room temperature or ○ complementary strands of RNA (RNA-RNA)
subject to refrigeration, you have to freeze them
DNA Base Pairs
● Adenine with Thymine (A with T)
Method Explanation Assessment ○ “Apple in the Tree”
(remember) ● Guanine with Cytosine (G with C)
○ “Car in the Garage”
Yield Spectrophotometry Nucleic acids DNA ug/mL:
absorb light at A260 x 50 ● The two participating populations of nucleic acids should
260 nm be SINGLE-STRANDED. Can only occur in nucleic
RNA ug/mL: acids.
A260 x 40
● The hybrid formed can be:
○ Stable - many areas possible for base pairing
Gel electrophoresis Intensity of Brighter bands =
○ Unstable - not many areas possible for base
or densitometry bands is higher yields
pairing
proportional to
**Depending on the extent to which complementary base pairing
takes place between the two strands.
1. SOUTHERN HYBRIDIZATION
● Also known as Southern Blot
● Named after Edwin Southern, who first reported the
procedure
● Allows the detection of a given DNA sequence in a
complex mixture of DNA sequences
SOUTHERN BLOT STEPS

1. Restriction Enzyme Digestion and Resolution
2. Preparation of Resolved DNA for Blotting (Transfer)
3. Blotting (Transfer)
4. Detection
- Hybridization - binding of the two complementary
strands
- Hybrid - product formed from the hybridization
BASIC COMPONENTS OF HYBRIDIZATION ASSAYS
1. PROBE
● A labeled molecule that binds specifically to the
Molecule of Interest
○ Can be fluorescently labeled when used in
hybridization
● Helps in the detection process
● e.g. Target Sequence (DNA/RNA)
a. RESTRICTION ENZYME DIGESTION AND
2. SAMPLE RESOLUTION
● Maintaining sample integrity is essential in hybridization ● Makes use of enzymes to digest the DNA
assays (especially RNA) molecules into small fragments
● Proper sample processing is important to maintain ● 10-50 ug of high-quality (intact) genomic DNA
nucleic acid integrity is required
● Immediate snap freezing and/or addition of lysis buffers ● Carried out for up to 3 hours to allow cutting of
allows sample (target) preservation (especially for RNA) all sites in the DNA sample
● Purification of the samples remove inhibitors and ● Fragments are resolved by gel electrophoresis
maximize accessibility of the target probe
○ Most recent procedures using commercial kits, - To fragment the DNA to detect whether a certain DNA
there is an important purification step that must sequence is present in that certain DNA fragment length
be performed so that inhibitors are removed
and that there is maximal accessibility of the b. PREPARATION OF RESOLVED DNA FOR BLOTTING
target group (TRANSFER)
3. CONTROLLED CONDITIONS PERMISSIVE FOR Denaturation

COMPLEMENTARY BASE PAIRING ● Process of separating the DNA fragments into
● Sensitivity and specificity of hybridization reactions rely single strands
on the optimum physical and chemical environment ● DNA is denatured by exposing the gel to a
during reaction strong base (example: NaOH)
● Hybridization cocktail: mixture of reagents selected to ● Effective on DNA fragments <500 bp
favor interaction of nucleic acids (mixture of probe,
nucleic acids, cofactors needed for hybridization assay); Depurination
this has many components and each should be ● Pre-denaturation process of larger DNA
optimized (concentration, volume, temperature) fragments (>500 bp)
● Gel is soaked in dilute HCl to remove the purine
4. CONTROLS bases from the sugar-phosphate backbone
● Each hybridization assay requires both positive and ● Loosens up the larger fragments for more
negative controls for validation complete denaturation
● Positive Sample Control: known to contain sequences - This is done before denaturation.
complementary to the probe
● Negative Sample Control: known not to contain c. BLOTTING (TRANSFER)
sequences complementary to the probe; used to monitor ● DNA fragments are transferred (single stranded
specificity; assess if you have nonspecific molecules) to a solid substrate that will facilitate
complementary binding of targets probe binding and signal detection
● Substrates:
○ Nitrocellulose
DNA Hybridization Techniques ○ Nylon
HYBRIDIZATION ASSAY FORMATS ○ Cellulose
1. Southern Hybridization (DNA)
2. Northern Hybridization (RNA) CAPILLARY TRANSFER
3. In Situ Hybridization ● Denatured DNA moves from gel to the membrane
4. Microarrays through the capillary movement of buffer from the
** Western blot is not included because it is not a soaked paper to the dry paper
hybridization technique, it is only for proteins and not for nucleic ● Advantages: simple, inexpensive, no instruments
acids. required
● Disadvantage: slow; less optimal transfer with large
gels; affected by bubbles, salt crystals
ELECTROPHORETIC TRANSFER
a. RNA EXTRACTION
● Uses electric current to move the DNA transversely
● RNA is isolated or extracted from clinical
through the gel to the membrane
samples in an RNAse-free environment (solid
● Used mostly for small fragments or proteins
phase, inorganic, organic)
b. ELECTROPHORESIS
● When you have isolated a pure RNA sample,
separate the RNA by their sizes using
electrophoresis.
c. NORTHERN BLOTTING
● Transfer of RNA to a membrane. The
membrane is similar to Southern Hybridization
VACUUM TRANSFER (e.g., nitrocellulose)
● Uses suction to move the DNA from the gel to the d. ADD LABEL
membrane in a recirculating buffer ● Add your label probes, if your target is present,
● Advantages: rapid transfer, avoids discontinuous your probes will emit signals and this will be
transfer (fastest among the recently mentioned visualized using x-ray films or other detection
procedures) techniques.
● Disadvantage: expense and maintenance of equipment
RNA EXTRACTION
● RNA is isolated or extracted from clinical samples in an
RNAse-free environment
d. DETECTION
● DNA fragments are exposed to a labeled probe
● Probes may be labeled with radioactive
phosphorus atoms
● The signal of the probe is detected to indicate
the presence or absence (lack of signal) of the 2. GEL ELECTROPHORESIS
sequence in question ● Purpose: To differentiate the RNA molecules by size
● One of the most important components of ● 0.8% to 1.5% of agarose gels are usually used (larger
Nucleic Acid Hybridization transcripts, remember the percentage)
● Polyacrylamide gels may be used especially for smaller
transcripts (those used for analysis of viral gene
expression; used for smaller transcripts since they are
less porous)
● Carried out under denaturing conditions for:
○ Accurate transcript size assessment
○ Efficient transfer of the RNA from the gel to the
membrane
● Representative lanes of the gel can be stained with
acridine orange or ethidium bromide (not commonly
used today since it is carcinogen) to assess quality and
equivalent sample loading
● Denaturants such as formaldehyde must be removed
from the gel before transfer because it inhibits binding of
the RNA to the nitrocellulose
2. NORTHERN HYBRIDIZATION
● Ammonium acetate or deionized water can be used to
● Also known as northern blot technique
remove the denaturant
● Modification of the Southern blot technique
● Designed to investigate RNA structure and quantity
BLOTTING (TRANSFER)
○ Levels of gene expression and stability
● RNA (is negatively charged, to avoid repelling) is
○ RNA structural abnormalities resulting from
transferred to a positively charged nylon or
synthesis and processing aberrations (e.g.,
nitrocellulose membrane and then immobilized for
alternative splicing)
subsequent hybridization
● Downward elution
○ best, low-tech method for agarose transfer;
○ results in tighter bands and more signal than
upward transfer
PRE-HYBRIDIZATION & HYBRIDIZATION WITH PROBE
● Prehybridization (blocking) prevents probe from ● The dotted material is arranged in a regular grid-like
coating the membrane and minimize background pattern.
problems (avoid premature binding) ● The sample is labeled and the arrayed features are
● Hybridization allows the probe to hybridize, or bind, to a referred to as probes.
specific RNA fragment on the membrane ● Allows genome-wide transcription profiling or
● Probes (small pieces of DNA or RNA) labeled with sequence determination in a single experiment
fluorescent dye or radioactive atom hybridize to the ○ Allows detection of many targets at the same
target RNA (to know if hybridization has occurred) time
● Probes are designed to have a sequence ○ Probes are unique from each other
complementary to a particular RNA sequence in the ○ Hybridization → presence of target
sample
DETECTION
● If a radiolabeled probe is used, the blot should be kept
from drying out and then immediately exposed to film for
autoradiography
● If a non-isotopic probe is used, the blot must be treated
with non-isotopic detection reagents prior to film
exposure
5. IN-SITU HYBRIDIZATION
● Involves taking morphologically intact tissue, cells, or
3. DOT/SLOT BLOTS
chromosomes affixed to a glass microscope slide
● Used to quickly analyze DNA and RNA without having to
through the hybridization process
consider the size of the target
● Methods of detection: autoradiography, colorimetry,
● Applications:
fluorescence
○ Expression
● Evaluation of final product is similar to evaluation of
○ Mutation
immunohistochemistry (ELISA-like but on a tissue
○ Amplification/deletion analyses
sample)
● Dot blots
○ target is deposited in a circle or dot
FISH VARIATIONS
○ useful for multiple qualitative analyses where
● Fluorescence In Situ Hybridization (FISH)
many targets are being compared (e.g.,
● Fast-FISH
mutational screening)
○ Used for quick hybridization, STAT cases
● Fluorescence Immunophenotyping and Interphase
Cytogenetics as a Tool for Investigation of Neoplasms
(FICTION)
● Fiber-FISH
Polymerase Chain Reaction

● Also considered as a nucleic acid hybridization reaction,
● Slot blots since you have to separate the DNA strands. Specifically,
○ Modification of dot blot a solution–based hybridization format as it happens in a
○ target is deposited in an oblong bar liquid format.
○ more accurate for quantification by
densitometry scanning
○ The area to which the target will be deposited is Principle
oblong instead of circle (difference in shape) ● Goal: To make multiple copies of DNA from a single copy
PCR Reagents
Polymerase Chain Reaction (PCR)

● In vitro synthesis of DNA
● “molecular photocopying”
REMINDERS ● Kary Mullis and Michael Smith: Nobel Prize in
● Probe hybridization should be optimized Chemistry (1993) for contributions to the developments
● Addition of negative control that will serve as the baseline of methods within DNA-based chemistry
for interpretation (to know if it is false positive or
negative)
● Include an amplification or normalization for loading or Components of PCR
sample differences correction ● DNA Template
● Primers (Forward & Reverse)
4. MICROARRAY ● Deoxynucleoside triphosphates (A, T, C, G)
● a.k.a DNA chip technology ● DNA Polymerase
● glass slide carrying hundreds to thousands of probes ● Mg2+ (MgCl or MgSO4)
● test samples: cDNA generated from sample RNA,
genomic DNA, or protein ● PCR Buffer
● Enhancers/additives
● Control (Positive and Negative)
DNA Template
● Derived from the patient’s genomic or mitochondrial
DNA, or from bacteria, fungi, or parasites
● Characteristics of a good DNA template:
○ Good condition
○ Free of contaminating protein
○ Without nicks or breaks
■ Will premature halt or stop the
synthesize of strands
○ With low GC content
■ The bond between guanine-cytosine DNA Polymerase
is really strong, making them hard to ● Critical key players in replicating the target DNA
separate ● Types:
○ DNA polymerase from E. coli: inactivated at
● Optimization of DNA input is important because higher
high temperatures
amounts of template increase the the risk of non-specific
○ Taq Polymerase: isolated from Thermus
amplification, whereas lower amounts reduce yields
aquaticus; heat-stable
● Plasmid DNA: 0.1– 1 ng
○ Tth Polymerase: isolated from Thermus
● gDNA: 5-50 ng
thermophilus with reverse transcriptase activity
● Increased amounts of DNA polymerase can help with
PCR yields, but may produce non-specific amplicons
○ Always optimize
○ Increased concentration - non-specific
amplicons, non-specific products, mis-priming
○ Decreased concentration - PCR will not
proceed, low yield of products
Primers
● Determine the specificity of PCR; contain sequences
complementary to sites flanking the region to be
analyzed
● Forward and Reverse Primers
Required Cofactor: Mg2+

● Magnesium ions function as cofactors for activity of DNA
polymerases
● Enabling the incorporation of dNTPs during
polymerization
● Catalyze phosphodiester bond formation between the
● Higher concentrations often contribute to mispriming 3’-OH of a primer and the phosphate group of a dNTP
and non-specific amplification
● Low primer concentrations can result in low or no
amplification of the desired product
Deoxynucleoside triphosphates (dNTPs) ● Low Mg2+ concentrations result in little to no PCR

● 4 nucleotides - adenine, thymine, guanine, cytosine product
● Act as “bricks” / building blocks ● High Mg2+ concentrations produce non-specific PCR
● Building blocks that extend the primer products (enhanced stability of primer-template
● Added to the PCR reaction in equimolar amounts for complexes and increase in replication errors from
optimal base incorporation misincorporation of dNTPs
● dNTPs exceeding optimal concentrations can inhibit PCR
● For efficient incorporation by DNA polymerase, free
dNTPs should be present in the reaction at a
concentration of no less than 0.010–0.015 mM
PCR Buffers
● Provide optimal conditions for polymerase enzyme
activity
● Buffer pH: 8.0 – 9.5, stabilized by Tris-HCl
● Examples: Potassium chloride, Ammonium sulfate
● The salts in these buffers affect denaturing and
annealing
● temperatures of the DNA and the enzyme activity
PCR Controls
PCR Run Amplification Steps
Control Description Purpose Expected
Reaction
Positive Contains Ensures that Target

control nucleic acid polymerase is sequence
sequence of active, primers & detected
interest thermocycler
working properly Denaturation
● Double-stranded template DNA is denatured into 2 single
Negative Contain all Checks for Target strands
control PCR contamination sequence not ○ Separation into 2 single strands
(contaminatio components with target DNA detected ● Heating the sample at 94-96 degrees Celsius
n control, except DNA or amplification
reagent from previous
blank, or no amplifications
target control)
Negative Contains DNA Ensures that Target

template but not target primers aren’t sequence not
control DNA binding to wrong detected
DNA
sequences.
Annealing
● 2 oligonucleotide primers anneal (hybridize) to
Internal Contains 2nd Demonstrates Housekeeping complementary sequences on the template
control set of primers that PCR is gene detected ● Annealing temperature must be optimized with the
(amplification and unrelated working. primers and reaction conditions (50-70 degrees Celsius)
control) target Differentiates
(housekeeping true neg from
gene or other false neg due to
nucleic acid amplification
target failure.
introduced in Detects PCR
sample before inhibitors.
nucleic acid
extraction or
during PCR
Primer Extension/Elongation
● DNA synthesis occurs
The PCR Workflow ● Polymerase synthesizes a copy of the template DNA by
Sample collection > DNA Isolation and Purification > DNA adding nucleotides to the hybridized primers
Quantification > Preparation of PCR Master Mix > PCR Run ● Occurs at 68-72 degrees Celsius
Amplification > Gel Electrophoresis > Gel Viewing and Data
Analysis
PCR Formats
METHOD EXPLANATION
PCR Modification
Chemical (Maxam-Gilbert) Sequencing
Nested PCR Uses 2 pairs primers and 2nd round of ● Developed by Allan Maxam and Walter Gilbert
amplification to increase sensitivity &
● Requires a double/single-stranded DNA fragment, with 1
specificity
end radioactively-labeled
● The labeled template is aliquoted into 4 tubes, each
Real-Time PCR Simultaneous amplification and detection
(qPCR) (fluorescence) in a sealed tube. Less treated with a different chemical with or without high salt.
subject to contamination. Eliminated
need for analysis of product by gel
electrophoresis. Quantitative. Commonly
used for detection of microorganism
Multiplex Uses more than 1 set of primers so

multiple targets can be amplified in same
tube
(Only one w/ only 1 round of
amplification)
● DMS: dimethylsulphate
Reverse Transcriptase Method to amplify RNA (mRNA or ● FA: formic acid
PCR microbial RNA). Same procedure as ● H: hydrazine
(RT-PCR) PCR except RNA is 1st converted to ● H+S: hydrazine + salt
cDNA by reverse transcriptase. Used to
● Specific Base Reactions in Maxam-Gilbert
detect HIV and HCV, measure viral loads
in HIV & HCV infections, measure gene Sequencing: Upon addition of a strong reducing agent
expression. (10% piperidine), single-stranded DNA breaks at
specific nucleotides.
Transcription-based Non-PCR methods to amplify target ● Fragments are separated by size on denaturing
amplification systems RNA. Similar methods, different polyacrylamide gel.
(TAS) manufacturers, cDNA is formed from ● The gel is read from the bottom to the top (5’-3’).
Self-sustaining target RNA. Millions of copies of RNA ● The size of the fragments gives the order of the
sequence replication produced by transcription of cDNA. nucleotides.
(3SR) Assays to detect RNA viruses.
● The lane in which a band appears identifies the
Transcription-mediated Mycobacterium tuberculosis, Chlamydia
amplification (TMA) trichomatis, TMA & NASBA are nucleotide.
Nucleic acid isothermal reactions. Don’t require ○ G and G+A lanes: Guanine (G)
sequence-based thermocycler. ○ C+T and C lanes: Cytosine (C)
amplification (NASBA) ○ C+T lane: Thymine (T)
○ G+A lane: Adenine (A)
● Downside: chemical sequencing
DNA Sequencing
● A laboratory technique that determines the exact

sequence or order or bases (A, C, G, and T) in a DNA
molecule
● DNA sequence information is important in investigating
the functions of genes, detecting mutations and genetic
variation, and determining polymorphism sites
Lecture Contents
A. Direct Sequencing
● Chemical (Maxam-Gilbert) Sequencing
● Dideoxy Chain Termination Sequencing Dideoxy Chain Termination Sequencing
B. Pyrosequencing ● a.k.a. Sanger Sequencing
C. Bisulfite DNA Sequencing ● Modification of the DNA replication process
D. Next Generation Sequencing ● Requires a single-stranded template and a
● Ion-Conductance Sequencing single-stranded primer covalently labeled with
● Reversible Dye Terminator Sequencing radioactive phosphorus or fluorescent dye
● Sequencing by Ligation
● Nanopore Sequencing
DIRECT SEQUENCING
● Direct sequencing involves reading the letters of the
genetic code directly. ● Modified dideoxynucleotide (ddNTP) derivatives are
● Most definitive molecular method to identify genetic added to the reaction mixture.
mutations or polymorphisms, especially when looking for ● ddNTPs lack the hydroxyl group found on the 3’ ribose
changes affecting only 1 or 2 nucleotides. carbon of the deoxynucleotides.
● Incorporation of ddNTP will terminate the replication ● Luciferase
process. ● Substrates
○ Adenosine 5’ phosphosulfate (APS)
○ Luciferin
● Components required for DNA synthesis (template,

Step 1:
primer, enzyme, buffers, dNTPs) are mixed with a
different ddNTP in each of 4 tubes
● The newly synthesized DNA strands will terminate at
each opportunity to incorporate a ddNTP
● Resulting synthesis products are a series of fragments ● one of the four dNTPs is added to the reaction with the
ending in either A (ddATP), C (ddCTP), G (ddGTP), or T template polymerase extends the primer; pyrophosphate
(ddTTP) (PPi) is released with the formation of the phosphodiester
bond between the dNTP and the primer
Step 2:
● released PPi is acts as a cofactor for ATP generation

from adenosine 5’ phosphosulfate
● Luciferase + ATP converts converts luciferin to
● The sets of synthesized fragments are loaded onto oxyluciferin
denaturing PAGE
● The four-lane gel electrophoresis pattern of the products Step 3:
of the four sequencing reactions is called a sequencing
ladder
● A sequencing ladder is read from the bottom to the top;
the smallest (fastest migrating) fragment represents the
first nucleotide attached to the primer
● Apyrase degrades residual free dNTP and dATP as

nucleotides are added on at a time, the sequence is
determined by which of the four nucleotides generates a
light signal
● Pyrogram shows the results from pyrosequencing

Pyrosequencing reactions; consists of peaks of luminescence associated
● Sequencing technique designed to determine a DNA with the addition of the complementary nucleotide
sequence without having to make a sequencing ● If the sequence contains a repeated nucleotide, the
ladder peak height doubles
● Relies on the generation of light (luminescence) from
when nucleotides are added to a growing strand of DNA
○ There is emission of light
● No gels, fluorescent dyes, and ddNTPs
○ Only based on lights
○ Superior compared to other techniques
Pyrosequencing Reaction Mix Components:

● Single-stranded DNA template
● Sequencing Primer Bisulfite DNA Sequencing
● Sulfurylase ● AKA methylation-specific sequencing
● Used to detect methylated cytosine nucleotides
● Methylation of cytosine residues to 5-methylcytosines in
DNA is an important part of regulating gene expression
and chromatin structure
● Incubation of DNA with bisulfite solution will deaminate
the cytosines (converted to uracils), whereas the
5-methylcytosines are unchanged
Steps:
● DNA fragmentation using restriction enzymes Reversible Dye Terminator Sequencing
● Agarose gel electrophoresis resolution of fragments and ● Captured or amplified fragments are hybridized to
purification immobilized primers on a solid surface (flow cell)
● DNA denaturation (97 degrees Celsius for 5 minutes) ● Fragments bind to the immobilized primers and are
● Bisulfite solution (sodium bisulfite, NaOH, hydroquinone) amplified by branch PCR to form collections of products
exposure called polonies
● Cleaning or precipitating of treated DNA ● Polonies are sequenced by the sequential addition of
● Polymerase chain reaction fluorescently labeled nucleotides
● Amplicon sequencing (Sanger or pyrosequencing)
● An image is taken of the flow cell after each nucleotide

addition, recording the presence of each nucleotide color
and location
● Fluorescent dyes are removed after imaging, and the
next nucleotide is added
Next Generation Sequencing
● Also called massive parallel sequencing
● Designed to sequence large numbers of templates
carrying millions of bases simultaneously
○ Less hassle to do than other formats of
sequencing
● Requirements:
○ Novel methods of template preparation
○ Powerful computer data assembly systems
○ Strong computer support and terabytes of
storage space
○ Interface with laboratory information systems Sequencing by Ligation
and electronic medical records ● Uses a pool of labeled oligonucleotide DNA ligase to
identify the template sequence through the known probe
Ion-Conductance Sequencing sequence
● The ion being referred to is “hydrogen ion”.
● Indexed libraries (collection of DNA fragments to be
sequenced) are amplified using primers immobilized on
microparticles (beads) in aqueous oil emulsion using
adapters on the library fragments complementary to the
immobilized primers
● Library amplification is performed in emulsion PCR
Nanopore Sequencing
● Advantage: does not require fragmentation and
● DNA polymerase will catalyze the formation of a
amplification of the template DNA
phosphodiester bond if the nucleotide is complementary
● Uses protein ion channels through which one strand of
to the sequencing template and a hydrogen ion is
DNA is drawn
released
● Each nucleotide is identified by a disruption in current
● Hydrogen ion lowers the pH of the reaction and
as it passes through the pore
recorded by the sequencer
○ There is a change in current as one nucleotide ■ Ovalbumin is the protein of egg
passes the pore. white, used as an amino acid source
for the developing embryo.
● Transport proteins
○ Purpose: transport of substances
○ Example: Haptoglobin, the iron-containing
protein of vertebrate blood, transport oxygen
from the lungs to other parts of the body.
■ Other protein transport molecules
across cell membranes.
● Hormonal proteins
○ Purpose: coordination of organism’s activities
Additonal information:
○ Example:Insulin, a hormone secreted by the
● Omicron variants are identified through sequencing. pancreas (islet of langerhans), causes other
tisues to take up glucose, thus, regulating blood
Proteins sugar concentration and other hormones.
● End product of transcription and translation: proteins ● Receptor proteins
● Most abundant biological macromolecules occurring in ○ Function: response of cell to chemical stimuli
all cells ○ Example: receptors built into the membrane of
○ All cells in the body are made up of protein. a nerve cell detects signalling molecules
● Most versatile organic molecule of the living systems released by other nerve cells.
and occur in great variety due to AA sequences forming ● Contractile and motor proteins (Actin and myosin)
certain proteins ○ Function: movement
○ Great variety - due to sequences of amino ○ Examples: motor proteins are responsible for
acids the undulations of cilia and myosin. Actin and
● Polymers of amino acids covalently linked by peptide myosin proteins are responsible for the
bonds contraction of muscles.
● Structural proteins
○ Function: support
■ To give support to the organs it
surrounds
○ Example: Keratin is the protein of hair, horns,
feathers, and other appendages. Insect and
spiders use silk fibers to make their cocoons
and webs respectively. Collagen and elastin
proteins provide a fibrous framework in animal
connective tissue.
Polypeptides
● Polymers of amino acids
● A protein is a biologically functionally molecule that
consists of one or more polypeptides
Amino Acid Monomers

● An amino acid is an organic molecule possessing both
an amino group and a carboxyl group
Protein Functions: ● The physical and chemical properties of the side chain
● Enzymatic proteins determine the unique characteristics of an amino acid
○ Purpose: For selective acceleration of ○ Side chain - determines the uniqueness
chemical reactions ● Groups of amino acids according to side chain
■ Hasten the reactions properties:
○ Examples: digestive enzymes - catalyzes the ○ Nonpolar side chains; hydrophobic
hydrolysis of bonds in foods ○ Polar side chains; hydrophilic
● Defensive proteins ○ Electrically charged side chains; hydrophilic
○ Purpose: protection against disease
○ Example: antibodies inactivate and help
destrroy virus and bacteria
■ Specifically, globulin
● Storage proteins
○ Purpose: storage of amino acids
○ Example: casein, the protein of milk, is the
major of amino acids for baby mammals. Plants
have storage proteins in their seeds.
Secondary Structure
● Regions stabilized by hydrogen bonds between atoms
of the polypeptide backbone
● Alpha helix: delicate coil held together by hydrogen
bonding between every fourth amino acid
● Beta pleated sheet: 2 or more strands of the
polypeptide chain lying side by side are connected
between parts of the two parallel polypeptide backbone
Amino Acid Polymers

● Composed of many amino acid monomers linked by a
peptide bond
Tertiary Structure
● 3-dimensional shape stabilized by interactions between
side chains
● Contributed by:
○ Hydrophobic interaction
○ Disulfide bridges
Structure and Function

Protein Structure and Function
● Frederick Sanger: pioneered the determination of the
amino acid sequence of proteins by working with the
hormone insulin Quaternary Structure
○ Also reponsible for Sanger sequencing ● Association of multiple polypeptides (foldings), forming a
● The protein’s structure determines its function functional protein
● 4 Levels of Protein Structure ● Overall protein structure resulting from the aggregation
○ Primary Structure (formation) of polypeptide subunits
○ Secondary Structure ○ Example: Transthyretin proteins: has 4
○ Tertiary Structure identical polypeptides
○ Quaternary Structure ○ Other examples: collagen and hemoglobin
■ Hemoglobin structure differs in
Primary Structure different age group
● Linear chain of amino acids
○ Order of amino acids
● Determined by inherited genetic information
● Dictates secondary and tertiary structure
● Cell disruption by enzymes that digest the cell wall
(especially, for plants and animal cells that have thicker
cell walls, same with yeasts and bacterial cells)
○ Cellulase and pectinase for plant cells
○ Lyticase for yeast cells
○ Lysozyme for bacterial cells - to acquire the
DNA
● Any variations in the amino acid may elicit different

phenotypic effect on an individual.
○ Examples:
■ In a patient with Sickle cell 2. Mechanical Rupture
hemoglobin / sickle cell anemia, ● Biological material with tough cell walls and many
the amino acid in the 6th position has tissue types require more rigorous methods
been replaced by valine instead of
glutamine. Examples:
■ Even a slight modification in a primary a. Sonication
structure can give an effect to its ● Cells in suspension, cooled on ice to avoid
overall phenotype. heating, are disrupted by shear forces using
short bursts of ultrasonic waves
Protein Preparation ○ Work on ice since this technique
● In order to obtain protein from a biological specimen, the generate a lot of heat or to prohibit
sample must first be harvested from the organism, the chance of protein degradation due
culture, or patient to heat
● Samples can be immediately frozen at -80 degrees
Celsius or processed fresh
○ Unlike DNA or RNA, isolating protein is less
hassle and requires less stricter protocols.
● Cell disruption is the first step of protein preparation
○ 2 classifications:
■ Gentle Cell Disruption
■ Mechanical Rupture (Harsher
method)
1.Gentle Cell Disruption

● Employed when the sample of interest consists of cells b. French Press
that lyse easily (i.e., RBCs and tissue culture cells) ● Cells are lysed by shear forces by forcing a cell
○ Avoid damaging proteins since these cells are suspension through a small orifice at high
delicate pressure
Examples: ○ Allow your cells to pass through
very small hole, then apply
a. Osmotic Lysis pressure
● Cells swell and burst when they are suspended
in hypotonic solution, releasing all cellular
contents
b. Freeze-Thaw Lysis
● Cells are lysed by subjecting them to one or
more cycles of quick freezing using liquid
nitrogen and subsequent thawing
c. Detergent Lysis
● Cellular membranes can be solubilized
(dissolve) just by suspending the cells in many
c. Mortar and Pestle
detergent-containing solutions
● Can be used to break down cells of solid
tissues and microorganisms
● The mortar is filled with liquid nitrogen and
the tissue or cells are grounded to a fine
powder
○ Liquid nitrogen allows immediate
freezing of samples
d. Mechanical Homogenization
● Employed by using handheld devices, blenders,
or other motorized devices (i.e vortex mixer)
Lysis by Isosmotic Solutions ● Glass bead homogenization can also be
employed for cell disruption
How to proceed with precipitation of proteins:
I. Acetone Precipitation - for water-soluble proteins
● Sequesters mostly water-soluble proteins
● Concentration-based procedure
○ Meaning: higher concentration of protein in
the original solution results in higher recovery
● Involves the addition of cold (0-20 degrees Celsius)
acetone to aqueous sample mixtures to a composition of
80%
Cell Lysis Conditions
Enzymatic Degradation During Cell Lysis

● Lysis must be done in a regulated manner as it also
causes degradation of enzymes, since they can alter the
composition of some substances.
● Lysis destroys the cell’s compartmentalization
resulting in the presence of hydrolases (e.g,
phosphatases, glycosidases, and proteases) in the
homogenate, altering the protein composition of the
lysed cells
● Enzymatic degradation must be avoided by placing the
freshly disrupted sample in solution with strong
denaturing agents such as 7-9 M urea, 2 M thiourea, or
2% SDS II. TCA Precipitation - for detergents
● Cell disruption is often performed at low temperatures ● Specific to deoxycholate and certain other detergents
to diminish enzyme activity in solution
○ To avoid enzymes to affect the composition of ○ Remove the detergents using TCA
proteins and to avoid them being activated, try ● In a typical TCA-deoxycholate precipitation, protein
to work on ice at all times solution is mixed with a dilute sodium deoxycholate
● Because enzymatic activity is pH dependent, unwanted buffer
hydrolysis is inhibited by lysing the protein sample at a ● Proteins are intercalated by deoxycholate and then
pH above 9 using sodium carbonate or tris precipitated from solution with trichloroacetic acid
(hydroxymethyl) aminomethane as a buffering agent (TCA)
○ pH above 9 - enzymes are no longer active ● Acidification of deoxycholate causes a change in
● Protease inhibitors are to be added to degrade solubility, causing aggregation and precipitation
proteases that are resistant to denaturation, pH, and ● Deoxycholate can be preferentially removed using
temperature change acetone
● Phosphatase inhibitors are necessary to block
phosphatases involved in phosphorylation pathways III. Methanol-Chloroform Precipitation
● Efficient method of precipitation and concentration for
PREPARATION OF CLEAN PROTEIN SAMPLES dilute samples, especially those containing membrane
proteins
Contaminant Removal by Precipitation ● Uses 4:1:3 ratio or methanol:chloroform:water, with
● Removal of contaminants (through precipitation) and additional 3 volumes of methanol to pellet the protein
detergents is necessary after cell lysis ● Proteins (slightly soluble in methanol-chloroform)
○ Interfere with enzymatic digestion accumulate on the water-organic interface and can be
○ Interfere with reverse-phase separations and pelleted by the additional methanol and centrifugation
mass spectrometry ● Used to concentrate dilute sample
○ Damage instruments and columns
● Removal of unwanted cellular material such as lipids and PROTEIN EXTRACTION FROM ARCHIVAL FFPE
genomic DNA can prevent chromatographic interference (Formalin-fixed Paraffin embedded) TISSUES
and it provides cleaner sample for downstream ● The use of fresh or frozen tissues for protein extraction
applications sometimes can be difficult, and results cannot
○ Because if you want to process proteins, you immediately be related to the clinical course of diseases
must have the cleanest protein sample you may ● Use of FFPE tissues for proteomics was thought to
have - done by removing contaminants be problematic
● The use of precipitation vary based on these factors: ○ Presence of formalin-induced protein cross
○ The detergent or contaminants for removal links and modification
■ Help to identify the course of action ■ Formalin coagulates proteins
you will employ ○ Deleterious effects of formalin-induced protein
○ Native or denatured state of proteins adducts and cross-links hampers proteomic
○ Post-processing analysis analysis
● Recent techniques proved that FFPE samples represent
an excellent resource for protein extraction
● Present tumor reaction > archived FFPE tissues > Principle of SDS-PAGE
microtome sectioning of FFPE tissue - deparaffinized and ● The use of SDS (also known as sodium lauryl sulfate)
stain with hematoxylin > Laser microdissection & and polyacrylamide gel eliminates the influence of the
collection of specific tumor cells - only collect the cells of structure and charge, and proteins are separated based
interest > Liquid tissue solubilization and trypsin on polypeptide chain length or their masses
digestion, to come up a peptide mixture > LC-MSMS ● SDS has a strong protein-denaturing effect
“shotgun” analyses – to identify the proteins ● In the presence of a reducing agent (2-mercaptoethanol),
disulfide bonds are reduced proteins unfold to linear
chains giving them a uniform negative charge
● SDS binding imparts a constant charge/mass ratio to the
protein
PROTEIN QUANTITATION
UV Spectroscopy Method: Detecting Proteins With Absorbance at
280 nm
- For DNA and RNA, read at 290 nm
● Amino acid residues with aromatic rings in a protein ● Polymerized polyacrylamide forms a mesh-like matrix for
absorb light the separation of proteins of typical sizes
● Tryptophan, tyrosine, and phenylalanine (TTP) ● Performed on a vertical electrophoresis set-up
contribute to the absorbance at 280 nm
● Concentration is computed based on the Beer-Lambert’s
Law
○ Concentration of a certain analyte is directly
proportional to absorbed light and it inversely
proportional to the transmitted light
● Only applicable for concentration determination on
protein sequences with known presence of tryptophan
and tyrosines (and phenylalanine)
UV Spectroscopy Measurement: Detecting Proteins with

Absorbance at 205 nm
● Based on absorbance by the peptide bond
● Used to quantitate protein solutions with concentrations
of 1-100 ug/mL protein Electrophoretic mobility depends upon 3 factors:
● More universally acceptable than A280 ● Shape: all proteins are in the primary structure after
● Lower concentrations of protein can be quantified with treatment with SDS and a denaturing agent; shape does
the A205 method not affect the protein separation
● Disadvantage: some buffers and other components ● Charge: all proteins become negatively charged after
absorb light at 205 nm treatment with SDS; charge does not affect the
separation
● Size: proteins get separated solely based on their
Techniques in Protein Analysis molecular weight
● SDS-PAGE
● Western Blot Functions of Reagents Used in SDS-PAGE
● MALDI-TOF ● 2-mercaptoethanol/dithiothreitol (DTT)
➢ breaks down disulfide bridges (S-S bridges)
SDS-PAGE present between the polypeptides of a protein
● SDS-PAGE: Sodium Dodecyl Sulfate – between cysteine residues
Polyacrylamide Gel Electrophoresis
● Analytical technique to separate proteins based on
their molecular weight or polypeptide chain length
● Involves the use of polyacrylamide as gel and SDS as
ionic detergent to denature and linearize proteins
● Developed by Ulrich Laemmli
● SDS ➢ Trailing Glycine molecules from Tris-Gly buffer
➢ imparts a negative charge to the protein ➢ Leading Chloride ions from Tris-HCl running
molecules evenly buffer
➢ It is a detergent
➢ when the voltage is applied, all protein migrate
towards the positive electrode of the gel
● Bromphenol Blue (BPB)

➢ works as a tracking dye in electrophoresis
➢ used to monitor progress of the molecules
moving through the gel
➢ Used to visually inspect the progress of your Running (Resolving) Gel
reaction ● Used to separate the polypeptides solely on the basis of
size
● Polyacrylamide Gel
➢ manufactured by the polymerization of
acrylamide in water by using a small amount of
a cross-linker
○ e.g. N,N’-Methylenebisacrylamide Major Steps of SDS-PAGE
➢ Acrylamide and bisacrylamide copolymerize 1. Pouring of the resolving/running and stacking gels
and create a mesh-like network 2. Loading the ladder and samples in wells
3. Running the gel by applying voltage
4. Subsequent analysis – Coomassie Blue Staining
Pouring of the resolving/running and stacking gels

● Gels are poured between two glass plates
● Bubbles are removed by adding a layer of isopropanol
● Stacking gel is loaded all the way to the top of the glass
plates; comb is placed after loading
Discontinuous Gels in SDS-PAGE

● The method developed by Laemmli makes use of
discontinuous gels
● “discontinuous” gels have stacking and running or
resolving gels with different compositions
Loading the ladder and samples in wells

● The ladder is added into the well on the extreme right
side using a micropipette
● The samples are added into the other well
● Note: do not puncture the wells
Stacking Gel
● Larger pores, lower acrylamide gel
● Stacks the mixture of proteins that needs to be separated
on the interface of stacking and resolving gel; all proteins
run into the separating gel at the same time no matter
what the size of the protein is
● Based on:
● Protease and phosphatase inhibitors are added to
prevent the digestion of the sample.
● Protein concentration is determined.
Gel Electrophoresis
● Commonly uses polyacrylamide gels and buffers loaded
with sodium dodecyl sulfate (SDS-PAGE)
● Types of gels:
➢ Stacking gel: used to concentrate all proteins
in one band
➢ Separating gel: allows for protein separation
Running the gel by applying voltage according to molecular weight
● Voltage is applied after dipping the “sandwich of gel and
glass plates” in running buffer
● Turn off the voltage when the tracking dye has reached
or crossed the gel
Protein Transfer
● Proteins are moved from within the gel onto a solid
Subsequent analysis – Coomassie Blue Staining support membrane to make the proteins accessible to
● Gel is rinsed with deionized water 3-5 times to remove antibody detection
SDS and buffer ● Electroblotting: uses an electric field to pull proteins out
● Gel is dipped in Coomassie Blue Stain of the gel and move into the membrane
● Invisible bands of proteins begin to appear within ● Done in semi-dry or wet conditions
minutes, but complete staining takes about 1 hour to
complete
WESTERN BLOT
● Introduced by Towbin et al. in 1979
● Commonly used method for qualitative and Blocking
semi-quantitative detection of proteins and post ● Prevent non-specific binding of antibodies to the
translational modifications; HIV diagnosis membrane, and does not reduce cross-reactivity
● Involves immunologic reaction ● Most commonly used blocking agents: Bovine serum
● Three elements: albumin (BSA) & non-fat dry milk
➢ Separation of proteins by size (SDS-PAGE) ● Blocking agents attach to all places in the membrane
➢ Transferring proteins to a solid support where the target proteins have not attached.
➢ Marking proteins by primary and secondary ● “noise” in the final product is reduced, resulting in
antibodies for visualization clearer results
Western Blot Procedure

1. Sample Preparation
2. Gel Electrophoresis (SDS-PAGE)
3. Protein Transfer
4. Blocking - prevent non specific binding of antibodies and
prevent cross-reactivity.
5. Antibody Incubation
6. Protein Detection and Visualization
Antibody Incubation
Sample Preparation ● The primary antibody binds to the target protein
● Washing the membrane with the antibody-buffer solution
● Proteins are extracted from different samples, such as helps in removing unbound antibodies
tissues or cells. ● The secondary antibody conjugated with an enzyme
is added based on the species of the primary antibody
MALDI Matrix Substance
Protein Detection and Visualization ● *This is not further discussed because it is too specific*
● A substrate reacts with the enzyme bound to the ● Functions:
secondary antibody for color development ○ Dilute and isolate analyte molecules from each
other
○ Mediator for energy absorption
● High polar analytes work better with highly polar
matrices, and non-polar analytes are preferably
Orange: Antigen combined with non-polar matrices
Blue: Primary Antibody ● Most commonly used matrices:
Red: Secondary Antibody ○ A-cyano-4-hydroxycinnamic acid
Green: Enzyme ○ 2,5-dihydroxybenzoic acid
Yellow: Substrate ○ 3,5-dimethoxy-4-hydroxycinnamic acid
○ 2,6-dihydroxyacetophenone
● Protein detection and visualization allows for the
determination of density and location of protein targets Time Of Flight (TOF) Analyzer
○ Western Blot - can be qualitative or ● Ions of different m/z are dispersed in time during their
quantitative determination. Needs to use a flight along a field-free drift path of known length
marker to estimate the size of proteins. ● Provided that all the ions start their journey at the same
● Size approximations are taken by comparing the protein time, the lighter ones will arrive earlier at the detector
bands to the marker than the heavier ones
● Detection systems: colorimetric, chemiluminescence, ○ Since, the lighter ones travel faster.
radioactive, fluorescence, electrochemiluminescence
MALDI-TOF Mass Spectrometry MALDI-TOF Mass Spectrometry Process

● Mass spectrometry: analytical technique in which
samples are ionized into charged molecules and their
mass-to-charge (m/z) ratio can be measured
● Matrix-assisted laser desorption/ionization (MALDI) is
the ion source
● Time-of-flight (TOF) analyzer is the mass analyzer.
● Not particularly used in the lab, but used in research.
Schematic diagram (Process overview):
1. Add matrix and sample for the ionization using MALDI
MALDI
2. A laser is hit for the ionization
● Soft ionization that involves a laser striking a matrix of
3. The ionized particles will go to the detector
small molecules to make the analyte molecules into the
4. Lighter ones will travel faster
gas phase.
5. MS Detector will identify your protein composition based
○ Purpose: ionize your material into its gaseous
on Mass spectrometry profile
phase.
6. Every protein will have its unique MS profile which will
● MALDI is appropriate to analyze biomolecules like
compared and identified using the MS profile
peptides, lipids, saccharides, or other organic
macromolecules
APPLICATIONS OF MALDI-TOF-MS
I. Intact Mass Determination
Principle of MALDI
II. Peptide mass Fingerprinting (MPF)
III. Oligonucleotide Analysis
IV. MALDI Imaging
I. Intact Mass Determination

● Important for protein characterization as the correct
molecular weight of a protein can indicate the intact
structure
● MALDI is suitable for fragile and easily fragmented
proteins
○ Because of ionization step
● MALDI-TOF is less affected by buffer components,
detergents, and contaminants
● MALDI-TOF permits intact protein mass determination
for sequence validation
○ Sequence of amino acids
II. Peptide Mass Fingerprinting

● Proteins are identified from simple mixtures by
MALDI-TOF coupled with two-dimensional gel
electrophoresis
● Peptides are generated by digesting proteins of interest
with a sequence-specific enzyme like trypsin
● Peptides are analyzed by MALDI-TOF to get the peptide ● In the DNA, the mutant sickle cell template strand (top)
has an A where the wild type (normal) template has a T.
masses ● The mutant mRNA has a U instead of an A in one codon.
● Peptide masses are compared against a database ● The mutant hemoglobin gas a valine (Val) instead of a
glutamic acid (Glu).
III. Oligonucleotide Analysis
● MALDI-TOF determines whether the synthesis of primers Take note:
and probes is complete and whether the synthesized ● Wild-type - Normal Phenotype
● Mutant - Abnormal/ Mutated Phenotype
sequence is correct
○ Counterchecking of synthesized primers using Types of Small-Scale Mutations
MALDI-TOF - Point mutations within a gene can be divided into two
● Oligonucleotide analysis using MALDI-TOF is a simple, general categories:
rapid, accurate, and sensitive method to determine 1. Single nucleotide-pair substitutions
complete oligonucleotide sequences 2. Nucleotide-pair insertions or deletions
1. Single Nucleotide-Pair Substitution

IV. MALDI Imaging
● Replacement of one nucleotide and its partner with
● MALDI-TOF can be used in profiling and imaging another pair of nucleotides
proteins directly from this tissue sections ● Some substitutions have no effect on the encoded
● Provides information about the local molecular protein
composition, relative abundance, and spatial distribution ● Example: If 3’-CCG-5’ on the template strand mutated to
of peptides and proteins 3’-CCA-5’, the mRNA codon would become GGU instead
of GGC. But, glycine would still be the amino acid
produced.
Additional Handout:
● GGU = Glycine, CCA = Glycine (no change in amino acid
produced) → SILENT MUTATION
GENETIC VARIATION AND MUTATION
Silent Mutation
Mutations ● Changes in a nucleotide pair, transforming one codon
● Changes in the nucleotide sequence of an organism’s into another that is translated into the same amino acid
DNA or in the DNA or RNA of a virus ● Has no observable effect on the phenotype
● Responsible for the huge diversity of genes found among ● Silent mutations can occur outside genes as well
organisms ● No effect on the protein
○ Not all mutations are bad.
■ Example: genetic modification
■ Mutations can result in genetic
diversity
■ Genetically modified food (GMO) -
confers resistance in crops against
pests
● Ultimate source of new genes
Major Types of Mutation

● Large-scale mutation
○ Chromosomal rearrangements affecting long Missense Mutations
segments of DNA ● Substitutions that change one amino acid to another one
● Small-scale mutation ● May have little effect on the protein
○ Variation of one or a few nucleotide pairs
○ Includes point mutations
■ Changes in a single nucleotide pair of
gene
○ Point mutation may be transmitted to the future
generations if it occurs on a gamete or in a cell
that gives rise to gametes
● If the mutation has an adverse effect on the phenotype

of an organism, the mutant condition is referred to as a
genetic disorder or hereditary disease.
○ Example: Sickle-cell hemoglobin Nonsense Mutation
● A point mutation that changes a codon for an amino acid ● Formats:
into a stop codon ○ Antigen detection: immobilization of
○ Stop codon: UGA, UAA, UAG antibodies to a solid matrix
● Causes translation to be terminated prematurely ○ Antibody detection: immobilization of antigens
● The resulting polypeptide will be shorter than the to detect antibodies
polypeptide encoded by the normal gene ● Example: ELISA
● Start codon: AUG
Example: Immunoassay format for the direct detection of antigens
2. Nucleotide Pair Insertions and Deletions
● Are additions or losses of nucleotide pairs in a gene Intensity of color will be proportional to the concentration of analyte
● Have a disastrous effect on the resulting protein more
often than substitutions do
● More damaging than substitution
Frameshift Mutation
● Occurs whenever the number of nucleotides inserted or
deleted is not a multiple of three
○ Reading frame: triplet codon
● Alters the reading frame of the genetic message, the
triplet grouping of nucleotides on the mRNA
Specific antibody Antigen binds Secondary Enzyme
to the target to the (detection) Abs substrate is
analyte is antibody. linked to an added that will
immobilized in a enzyme (e.g., generate
solid matrix (e.g., horseradish signals for
microtitre plate peroxidase, detection.
wells) alkaline
phosphatase) are
added.
2. Immunohistochemistry
● Perform on tissues, still dependent on antigen-antibody
complexes.
● Performed on thin (<5 micron) slices of fixed or 5 to

15-micron slices of frozen tissue
● Steps:
DETECTION OF GENE MUTATIONS 1. Deparaffinization (for paraffin-embedded tissue
samples) and rehydration of tissue sample
Biochemical Methods 2. Antigen retrieval to expose epitopes
● Used to detect or quantify the altered protein product, (antibody-binding sites)
not the variation itself 3. Blocking with hydrogen peroxide, UV light, or
● Directly analyze the change in protein structure or 1% serum to prevent background signals
function rather than to search for potential point 4. Addition of antibody
mutations 5. Addition of substrate
● Commonly used methods: 6. Imaging or microscopic examination
1. Enzyme Immunoassays
2. Immunohistochemistry ● Provides the advantage of integrating target detection,
3. Automated methods (GC, HPLC, MS) localization, and quantification in the context of tissue
morphology
1. Enzyme Immunoassays ● Allows the detection of protein abnormalities in situ; the
● Involve the use of specific antibodies or other ligands to tissue and intracellular location of mutant proteins (or
detect the presence of target molecules lack thereof) can be observed
● Antibodies or other ligands are labeled with enzymes
that bind to their specific substrate for signal detection 3.1. High-Performance Liquid Chromatography
● Flexible, high-throughput
● Separation technique based on the distribution of the ● Wild type (normal) and mutant DNA samples display
analyte (sample) between a mobile phase (eluent) and different electrophoretic band patterns, visulaizing this
a stationary phase (packing material or column) using electrophoresis
● 2 major component: mobile phase and stationary phase
Visualization Methods (Overview):
1. Radioisotope labeling
2. Silver staining
3. Fluorescent dye-labeled PCR primers
4. Capillary-based electrophoresis
3.2. Gas Chromatography

● Separation of vaporized sample through a column of
inert carrier gas (mobile phase) and a high-boiling-point
liquid (stationary phase) that differentially adsorbs
molecules
● Strength of the interaction of the sample and liquid phase
will result in varying retention times
● Detection of drugs, poisons and their metabolites
● Reference laboratory: quantification of trace elements
and drugs → uses gas chromatography in tandem with Silver Staining SSCP analysis for detection of Threonine 594
mass spectrophotometry (GC-MS) Methionine mutation
● TM: heterozygous mutant
● TT: wild type (normal)
● MM: homozygous mutant
3.3. Mass Spectrometry

● Converts molecules to ions that can be moved in a
magnetic field based on their charge (z) and mass (m)
● The read-out of the instrument is a spectrum with the
mass/charge value on the x-axis and the abundance of
the ion on the y-axis
● Molecules can be identified by their characteristic
spectrum or set of peaks II. Allele-Specific Oligomer (ASO) Hybridization
○ Similar to time of flight ● Utilizes ASOs which are short piece of synthetic DNA
sequence complementary to the target DNA
● An ASO can act as a probe in Southern Blot or
Northern Blot Assays
● Makes use of two probes
○ One probe hybridizes to the wild-type DNA
sequence (normal)
○ Another probe hybridizes to the mutant
sequence
○ Match → hybridization
○ Mismatch → No hybridization
Nucleic Acid Analyses

● Classical molecular methodology of mutation detection
● DNA sequencing, although the most definitive method for
detecting mutations, may not be appropriate especially
for high-throughput procedures and is expensive
I. Single-Strand Conformation Polymorphism (SSCP)

● Single-stranded DNA (ssDNA) has a defined
conformation; altered conformation due to a single
change on the sequence can cause ssDNA to migrate
differently III. Restriction Fragment Length Polymorphism (RFLP)
● Pronounced as “rif-flip”
● Based on the specificity of restriction endonucleases,
which recognize a set of nucleotides at the restriction VIDEO DISCUSSIONS
site, and cleave the DNA at those sites
● Applications: PART 1
○ Genotyping of mutations
○ DNA fingerprinting
○ Mapping of genes
○ Diagnosis of genetic disorders
● Been used for years in the identification of certain genetic
variations and mutations
PART 2
Allelic Discrimination with Fluorogenic Probes

● Performed on thermal cyclers with fluorescent detection
support (e.g., real-time PCR) PART 3
● Makes use of 2 fluorescently-labeled probes that are
complementary to either the normal or mutant sequence
● The presence of the corresponding fluorescent signal
indicates whether the sequence is normal or mutant
● Common fluorescent dyes used: VICTM and FAMTM
● Example: TaqMan probe that detects the normal
sequence is labeled with FAMTM; while the probe that
detects the mutant sequence is labeled with VICTM

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MOLECULAR BIOLOGY W E E K｜21

MODULE WRITER: JEFFERYL KAE PANDAC MTAP 2

OUTLINE ● Institutional and personal security measures designed to

Biological Agent Biosafety vs. Biosecurity Issue

3. Increasing Levels of Protection Human Blood and Blood Products

Color Coding System of Waste Segregation

Rule of thumb in dealing with segregation errors

Other Waste Treatments

Function Carries genetic Converts genetic

Location Nucleus (except Nucleus and cytoplasm

Composition Repeating Repeating nucleotides

Ribosomal RNA (rRNA)

White K2EDTA & gel Isolation of plasma. Gel forms

Blue/Black Sodium Isolation of mononuclear cells.

Yellow Acid Citrate Enhanced WBC recovery for

DNA ISOLATION CHEMISTRIES

1. Organic Isolation Methods

General Scheme of Organic DNA Isolation

Rapid Extraction Methods

Guidelines to Observe when Handling RNA

Total RNA Composition Process:

Gel electrophoresis Position of DNA:

Nucleic Acid Hybridization

SOUTHERN BLOT STEPS

BASIC COMPONENTS OF HYBRIDIZATION ASSAYS

3. CONTROLLED CONDITIONS PERMISSIVE FOR Denaturation

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR)

Required Cofactor: Mg2+

Deoxynucleoside triphosphates (dNTPs) ● Low Mg2+ concentrations result in little to no PCR

Positive Contains Ensures that Target

Negative Contains DNA Ensures that Target

Multiplex Uses more than 1 set of primers so

● A laboratory technique that determines the exact

● Components required for DNA synthesis (template,

● released PPi is acts as a cofactor for ATP generation

● Apyrase degrades residual free dNTP and dATP as

● Pyrogram shows the results from pyrosequencing

Pyrosequencing Reaction Mix Components:

● An image is taken of the flow cell after each nucleotide

​Amino Acid Monomers

Amino Acid Polymers

Structure and Function

● Any variations in the amino acid may elicit different

1.Gentle Cell Disruption

Enzymatic Degradation During Cell Lysis

UV Spectroscopy Measurement: Detecting Proteins with

● Bromphenol Blue (BPB)

Pouring of the resolving/running and stacking gels

Discontinuous Gels in SDS-PAGE

Loading the ladder and samples in wells

Western Blot Procedure

MALDI-TOF Mass Spectrometry MALDI-TOF Mass Spectrometry Process

I. Intact Mass Determination

II. Peptide Mass Fingerprinting

1. Single Nucleotide-Pair Substitution

Major Types of Mutation

● If the mutation has an adverse effect on the phenotype

● Performed on thin (<5 micron) slices of fixed or 5 to

3.2. Gas Chromatography

3.3. Mass Spectrometry

Nucleic Acid Analyses

I. Single-Strand Conformation Polymorphism (SSCP)

Amino Acid Monomers