MLS 117 | MOLECULAR BIOLOGY LECTURE | MIDTERM

POLYMERASE CHAIN REACTION Taq polymerase

➔ the DNA polymerase typically used in PCR
➔ after the heat-tolerant bacterium from which it was
isolated (Thermus aquaticus)

Thermus aquaticus
➔ lives in hot springs and hydrothermal vents
➔ very heat-stable and is most active around 70°C
➔ this heat-stability makes Taq polymerase ideal
Polymerase Chain Reaction (PCR) for PCR.
Keypoints:
➔ is a technique to make many copies of a specific
DNA region (DNA template) in vitro
➔ relies on a thermostable DNA polymerase, Taq
polymerase, and requires DNA primers designed
specifically for the DNA region of interest.
➔ In PCR, the reaction is repeatedly cycled through a
series of temperature changes, which allow many
copies of the target region to be produced.
➔ Discovered around 1983 by Kary Mullis

PCR Primers
Uses:
➔ a short sequence (ssDNA) of nucleotides that
➔ Diagnosis of infections
provides a starting point for DNA synthesis.
➔ Crime investigations
➔ ~20 nucleotides in length
➔ Genetic research
➔ 2 primers are used in each PCR reaction

➔ a common laboratory technique used to make many

copies (millions or billions) of a particular region of
DNA.
➔ Typically, the goal of PCR is to make enough of the
target DNA region that it can be analyzed or used in
some other way. For instance, DNA amplified by PCR
may be sent for sequencing, visualized by gel
electrophoresis or cloned into a plasmid for further
experiments

PCR Components:
➔ DNA Template
➔ DNA Polymerase
➔ Oligonucleotide Primers
➔ Deoxyribonucleotide triphosphate
➔ Buffer System

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examining it next to a standard "yardstick" made
up of DNA fragments of known sizes.

Gel
➔ is a matrix of agarose molecules that are held
together by hydrogen bonds and form tiny pores.
➔ At one end, the gel has pocket-like indentations
called wells, which are where the DNA samples will
be placed.

Stages in the PCR

➔ Before the DNA samples are added, the gel must be

placed in a gel box. One end of the box is hooked to
a positive electrode, while the other end is hooked
to a negative electrode. The main body of the box,
where the gel is placed, is filled with a
salt-containing buffer solution that can conduct
current.

GEL ELECTROPHORESIS
➔ is a technique used to separate DNA fragments
based on their size and charge.
◆ electro = using electricity to make
molecules migrate
◆ phoresis = "migration" or "movement." ➔ One well is reserved for a DNA ladder, a standard
reference that contains DNA fragments of known
lengths.
Applications
➔ A typical voltage for running an agarose DNA gel
➔ All DNA molecules have the same amount of
would be in the range of 80–120 V.
charge per mass. Because of this, gel
◆ A higher voltage will make the gel run
electrophoresis of DNA fragments separates them
faster but may also melt it if it runs for a
based on size only.
long period of time.
➔ see how many different DNA fragments are present
◆ A lower voltage will make the gel run
in a sample and how large they are relative to one
more slowly
another.
➔ determine the absolute size of a piece of DNA by

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Visualizing the DNA Fragments

Band
➔ A well-defined “line” of DNA on a gel
➔ Each band contains a large number of DNA
fragments of the same size that have all traveled as a
group to the same position. A single DNA fragment
(or even a small group of DNA fragments) would not
be visible by itself on a gel.

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NUCLEIC ACID EXTRACTION
➔ WBCs, particularly polymorphonuclear leukocytes
(PMNs) and macrophages are used as samples.
➔ The first step in to perform in an extraction to
obtain a lysate (cell lysis)
◆ RNA extraction: lyses the cell to release
the RNA
◆ Without cell lysis, nucleic acids will not
be released
DNA
➔ made up of two strands of long chains of individual
nucleic acid molecules or nucleotides wound around
each other in a helix.
➔ DNA was a perfect molecule for storing, and copying
vast amounts of information – making it ideally
suited to store the “blueprints” for cells, and
therefore life forms.
➔ Blueprint for the genetic material
RNA
➔ polymeric molecule that is present in all biological
cells.
➔ principally involved in the synthesis of proteins,
coding, decoding, regulation & expression of genes.
➔ It carries the messenger instructions from DNA,
which itself contains the genetic instructions
required for the development and maintenance of
life
Significance of DNA/RNA Extractions
➔ Able to understand the physiology & metabolism of
the cell in molecular level
➔ Allows discovery of disease-related DNA sequences
including cancer, genetic disorders and microbial
infection
➔ Became the backbone of genetic engineering
➔ Extraction: DNA is obtained from the interior of the
cell; to obtain a pure sample of DNA or RNA
◆ Can be subjected to electrophoresis or
PCR
◆ Electrophoresis: the double stranded
DNA is placed on the wells to know the
number of base pairs
Purpose
➔ To release nucleic acid from the cell for use in other
procedures
◆ Nucleic acid should be PURE
◆ other contaminants such as proteins
should be removed
● proteinase: remove proteins
● surfactants: remove lipids
➔ Must be free from contamination with protein,
carbohydrate, lipids or other nucleic acids.
➔ Used pure nucleic acids for testing.
Isolation of DNA
➔ Routinely isolated from human, fungal, bacterial and
viral sources.
➔ Specimen for Processing:
◆ Whole blood
◆ Tissue samples
◆ Microorganisms
➔ Need sufficient sample for adequate yield.
➔ through exposure to Tris, the cell membrane
becomes permeable; hence, the cell can easily be
lysed
◆ DNAse enzyme: present in the cell that
degrades the DNA
● EDTA is used to block the
DNAse enzyme function
Isolation of DNA
Pretreatment
➔ NOTE: purpose of pretreatment: faster lysis
➔ Whole Blood – separate cells (WBC from RBC)
◆ Differential density gradient
centrifugation:
(Specimen + isotonic saline is overlaid with Ficoll)
◆ Differential lysis:
(Specimen + Hypotonic buffer or water = cell
lysis) then centrifuge to collect WBC in pellets
➔ Ficoll – density gradient medium, made of highly
branched polymer formed by the copolymerization
of sucrose and epichlorohydrin
◆ Ficoll reagent: harvests the
macrophages and WBCs
➔ TISSUE SAMPLE – from fresh / frozen /
fixed-embedded
◆ Fresh / Frozen Tissue
(Grinding in liquid nitrogen,
homogenizing the tissue, or simply
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mincing the tissue using a scalpel can
disrupt whole tissue samples)
◆ Fixed-Embedded Tissue:
(deparaffinized by soaking in xylene,
Histosolve, Anatech Pro-Par or
ParaClear. After xylene treatment, the
tissue is usually rehydrated by soaking it
in decreasing concentrations of ethanol.)
➔ Xylene: clearing agent
➔ Fresh sample: can be minced or grinded
➔ MICROORGANISMS: Bacteria / Fungi
◆ Cell wall digestion by enzymes
(lyzozyme or zymolyase)
◆ Mechanical method: grinding or by
vigorously mixing with glass beads
◆ Treatment with detergent (1% sodium
dodecyl sulfate) and strong base (0.2M
NaOH) in the presence of Tris base,
EDTA, and glucose can also break
bacterial cell walls.
◆ Boiling in 8% sucrose, 8% Triton X-100
detergent, Tris buffer, and EDTA after
lysozyme treatment releases DNA that
can be immediately precipitated with
alcohol.
➔ Note that even the shedding of skin may cause
contamination and lead to an erroneous result
➔ Function of SDS: anionic detergent that destroys
cell membrane
➔ Proteinase-K: destroys proteins in the cell; used
in DNA extraction
➔ Phenol/Chloroform – commonly used
DNA Isolation
➔ Must purify DNA by removing contaminants.
➔ Accomplished by using combination of high salt, low
pH and an organic mixture of phenol and
chloroform.
➔ To avoid RNA contamination, add RNAse
DNA Isolation
1. Organic Method: Phenol/Chloroform
2. Inorganic Method
3. Solid Phase Isolation
4. Crude Lysis Method
1. Organic Method: Phenol-Chloroform Extraction
➔ Biphasic emulsion forms
◆ Hydrophobic layer on bottom has cell
debris (pellet)
● Bottom: hydrophobic
◆ Hydrophilic layer on top has dissolved
DNA (supernatant)
● Top: aqueous (hydrophilic)
➔ Note that DNA due to its negative charge is more
polar; dissolves in the hydrophilic layer (supernatant)
➔ Uses SDS and Proteinase K for the enzymatic
digestion of proteins and nonnucleic acid cellular
components.
➔ A mixture of phenol:chloroform:isoamyl alcohol
(25:24:1) is then added to promote the partitioning of
lipids and cellular debris into the organic phase,
leaving isolated DNA in the aqueous phase.
➔ Following centrifugation, the aqueous phase
containing the purified DNA can be transferred to a
clean tube for analysis.
◆ Chloroform protects the DNA from lysis
◆ Proteinase K destroys the histones,
thus, freeing the DNA
● This is not used in RNA
extraction due to the absence of
histones
◆ Lysate solution: after the addition of
SDS and proteinase K which would
cause lysis
● Contaminants settle at the
bottom
◆ Ethanol/isopropanol, or high
concentration of salts: aids in
precipitating DNA
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CHLOROFORM
➔ Protect the genomic DNA
➔ increases the efficiency of phenol to denature
the protein.
➔ chloroform denatures the lipid as well.
ISOAMYL ALCOHOL
➔ helps in reducing foaming between interphase.
It prevents the emulsification of a solution.
◆ Isoamyl alcohol: non-foaming agent;
prevents emulsification of 2 solutions

1. Collect supernatant, add cold ethanol, DNA precipitates

out.
➔ Or isopropanol in a high concentration of salt
(ammonium, potassium or sodium acetate, or
lithium or sodium chloride)
2. Ethyl or isopropyl alcohol is added to the upper phase
solution at 2:1 or 1:1 ratios, then centrifuge
3. Excess salt is removed by rinsing the pellet in 70%
ethanol,
4. Centrifuge and discard the ethanol supernatant
5. Dissolve the DNA pellet in rehydration buffer, usually 10
mM Tris, 1 mM EDTA (TE), or water.

2. Inorganic Method: “Salting out”

➔ a simple and nontoxic DNA extraction technique,
introduced by Miller et al., that isolates a
high-quality DNA from the whole blood
➔ Less toxic; no need for fume hood
➔ Saturated brine solution (uses saturated salt
solution)
◆ Ammonium sulfate may also be used

PHENOL
➔ DNA is insoluble in phenol because phenol is a
less-polar solution.
➔ phenol unfolds the protein structure and
digests it.
➔ Phenol: non-polar; cannot dissolve DNA
◆ Toxic substance; should be performed
in the fume hood

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➔ Uses low pH and high salt condition to selectively
precipitate proteins (most common salt: ammonium
sulfate)
1. DNA is left in solution
2. Precipitate out DNA with isoproproanol
➔ Precipitated out DNA is usually referred to as
“snotty”
3. Resuspended in TE buffer or water.
➔ Tris-EDTA (TE) buffer solution - pH 8.0
3. Solid Phase Isolation
➔ More rapid and effective
◆ Use solid matrix to bind the DNA (Spin
Column)
◆ Wash away contaminants.
◆ Elute DNA from column
➔ Proteolytic Lysis of Fixed Material
◆ dewaxed with xylene or other agents
and then rehydrated before nucleic acid
isolation
➔ Microdissection
◆ stained serial section can be examined
microscopically to identify tumor cells
◆ The identifiable areas of tumor can then
be isolated directly from the slide by
simple scraping in buffer or laser
capture and deposited into
microcentrifuge tubes.
➔ Extraction With Chelating Resin:
◆ Chelex is a cation-chelating resin that
can be used for simple extraction of DNA
➔ Rapid lysis methods
➔ DNA extraction/storage cards

➔ Uses solid matrix to bind DNA (spin column)

◆ Nucleospin
➔ Elution: addition of RNAse/DNAse free-water;
removes the RNA/DNA from the silica membrane
➔ Centrifugation: washing procedure

4. Crude Lysis Method

Uses:
➔ Screening large numbers of samples
➔ Isolation of DNA in limited amounts
➔ Isolation from challenging samples, i.e., paraffin
embedded tissue
◆ Usually not done in clinical laboratory.

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RNA Isolation
➔ Requires STRICT precautions to avoid sample
degradation
◆ RNA = labile
➔ Note that RNA is very labile; requires strict
procedures
◆ This is to avoid sample degradation

Total RNA
➔ 80–90% of total RNA is ribosomal RNA.
➔ 2.5–5% is messenger RNA

RNAse
➔ are naturally occurring enzymes that degrade RNA
➔ Common laboratory contaminant
➔ Also released from cellular compartments during
isolation of RNA from biological samples
➔ Can be difficult to inactivate
➔ MUST be eliminated or inactivated BEFORE isolation
➔ CRITICAL to have a separate RNAse free area of lab.
➔ RNAse is a common laboratory contaminant because
this may also be found in the gloves
◆ Nitrile gloves should be used

Protecting against RNAse

➔ Wear gloves at all times
➔ Purpose of ethanol: so the RNA will bind in the
➔ Use RNase-free tubes and pipet tips
column with silica membrane
➔ Use dedicated, RNase-free, chemicals
➔ Ideally, gloves should be changed before adding
➔ Pre-treat materials with extended heat (180 C for
ethanol to the lysate
several hours), wash with DEPC-treated water,
NaOH or H2O2
➔ Supplement reactions with RNase inhibitors

RNA Isolation

NucleoSpin® Dx Virus
➔ is a generic kit which can easily be integrated into
in-vitro diagnostic workflows.
➔ The kit allows the purification of nucleic acid from
different viruses and can be used in combination
with a broad range of downstream assay systems.
➔ The system is suitable for fresh and frozen samples
treated with EDTA or citrate.