Tools used in Molecular Biology

Gel Electrophoresis

Gel electrophoresis is a procedure for separating

a mixture of molecules through a stationary
material (gel) in an electrical field.

Technique used for the separation, detection

and sometimes purification of biomolecules
[deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), or protein molecules] using an electric
field applied to a gel matrix.
 Gel Electrophoresis

Molecules are separated on the basis of size

and charge.

Bigger size molecules will move slower than the

molecules smaller in size in the porous gel matrix
 Gel Electrophoresis

• When charged molecules are placed in an electric field, they

migrate toward either the positive or negative pole
according to their charge.

• Proteins, which can have either a net positive or net

negative charge depending on the pH of solvent will move
towards anode/cathode.

• Nucleic acids have a consistent negative charge imparted by

their phosphate backbone, and migrate towards the anode.
 DNA
• Organic molecules such as DNA are charged. DNA is
negatively charged because the phosphates (red circles) that
form the sugar-phosphate backbone of a DNA molecule
have a negative charge.
 Gel electrophoresis matrix or "gel"

1. A gel is prepared which will act as a

support for separation of the
fragments of DNA.
2. The gel is a jelly-like material
3. Agarose is used for DNA
4. Holes are created in the gel.
5. These will serve as a reservoir to
hold the DNA solution.
6. The gel is immersed within an
electrophoresis buffer that provides
ions to carry a current and some
type of buffer to maintain the pH at a
relatively constant value.
DNA solutions
(mixtures of
different sizes
of DNA
fragments)
are loaded in
a well in the
gel.
•The gel matrix acts
as a sieve for DNA
molecules.

•Large molecules
have difficulty
getting through the
holes in the matrix.

•Small molecules
move easily through
the holes

•Because of this,
large fragments will
lag behind small
fragments as DNAs
migrate through the
gel.
As the
separation
process
continues, the
separation
between the
larger and
smaller
fragments
increases.
•Molecular weight
markers are often DNA ladder
electrophoresed
with DNAs. 750 bp
650 bp
•Molecular weight 550 bp
450 bp
markers are 350 bp
usually a mixture 300 bp
of DNAs with 200 bp
100 bp
known molecular
weights

•Molecular weight
markers are used
to estimate the
sizes of DNA
fragments in your
DNA sample
The actual process
of DNA
electrophoresis is
shown in the next
few slides.

The first step is to

prepare a tray to
hold the gel matrix
(agarose). The ends
of the tray are taped.
A "gel comb" is used to create holes in the gel.
The gel comb is placed in the tray.
1. Agarose powder
is mixed with a
buffer solution,
usually tris
borate EDTA
(TBE buffer).

2. The solution is
heated until the
agarose is
dissolved.

3. The hot agarose

solution is
poured into the
tray and allowed
to cool.
 After the gel is cooled, tape is
removed from the ends of the gel
tray and the gel tray is placed in an
electrophoresis chamber.

The electrophoresis chamber is

filled with buffer, covering the gel.
This allows electrical current from
poles at either end of the gel to flow

 
through the gel.

Finally, DNA samples are mixed                              

with a "loading dye". The loading
dye allows you to see the DNA as
you load it and contains glycerol or
sucrose to make the DNA sample
heavy so that it will sink to the
bottom of the well.
A safety cover is placed over
the gel (to keep you from
frying yourself) and
electrodes are attached to a
power supply.

Electrical current is applied.

DNA fragments will migrate

through the gel at various
rates, depending on their
size.

When the dye marker

indicates that DNA
fragments have moved
through the gel, the current
is turned off and the gel is
removed from the tray.
 Visualization of DNA bands
DNAs are visualized by
staining the gel with ethidium
bromide

EtBr will bind to DNA and will

fluoresce in UV light. The gel with UV illumination, the
ethidium bromide stained DNA glows

 
This photograph is of DNA orange

that has been

electrophoresed.

Note that high molecular

weight                              
DNAs do not separate well on
this gel.

This can be corrected by

altering gel density. (decrease
concentration of agarose) Digital photo of the gel.
(Used: 0.8% to 2.0% agarose)
Gel electrophoresis matrix or "gel"
 DNA/RNA purification from a mixture
1. Separate the DNAs in Agarose gel
2. Cut the required (DNA + Gel) band
3. Extract the DNA from gel
 Spectroscopy is the study of the interaction of
electromagnetic radiation in all its forms with
matter. 

UV-Vis spectroscopy and Fluorescence Spectroscopy commonly used in MolBio

 Method to visualize biomolecules (DNA)
Ethidium bromide (EtBr)
• Intercalates between the base pairs
• More hydrophobic environment on binding to DNA
• Shows fluorescence in UV light on binding to DNA

300nm Ex; 590nm Em

EtBr carcinogenic
Method to visualize biomolecules (DNA)
SYBR Green (safe)

SYBR stained DNA Gel,

480nm ex; 520nm Em

 Method to visualize biomolecules
Radioisotopes and Autoradiography
P32 for DNA/S35 for Proteins

Autoradiography: A technique using X- ray film to

visualize molecules or fragments of molecules that
have been radioactively labeled.

An autoradiograph is an image on an x-ray film or

nuclear emulsion produced by the pattern of decay
emissions (e.g., beta particles or gamma rays) from a
distribution of a radioactive substance.
Quantitative analysis by Densitometer
 Phosphorimager

A phosphor, generally, is a substance that exhibits the phenomenon of luminescence.

Luminescence (Molecule emitting light at normal temp or without heat)
Photostimulable Luminescence
Phosphorimager
 What is the difference between
fluorescence and phosphorescence?

What is Autoragiography or
Autoradiogram?

What is Luminescence?

What is phosphorimaging or
Phosphoimager?
 Gel Matrix

Gel is a cross linked polymer

1. Agarose gels

2. Polyacrylamide gels
 Agarose gels
Used for Nucleic acid separation and purification
• Agarose is a polysaccharide extracted from seaweed(Algae).
• It is typically used at concentrations of 0.5 to 2%.
• The higher the agarose concentration the "stiffer" the gel (low
molecular weight/smaller size separated).
• Agarose gels are extremely easy to prepare: you simply mix agarose
powder with buffer solution
• Melt it by heating,
• Pour the gel.
• It is also non-toxic. However, EtBr is carcinogen/Mutagen (SYBR Green
safer: fluorescent DNA binding dye)
• Agarose gels have a large range of separation, but relatively low
resolving power.
• By varying the concentration of agarose, fragments of DNA from about
200 to 50,000 bp can be separated using standard electrophoretic
techniques.
 Polyacrylamide
1. Polyacrylamide is a cross-linked polymer of acrylamide.

2. The length of the polymer chains is dictated by the

concentration of acrylamide used, which is typically between
3.5 and 20%.
3. Acrylamide is a potent neurotoxin and should be handled with
care! Wear disposable gloves when handling solutions of
acrylamide, and a mask when weighing out powder.
4. Gels should also be handled with gloves due to the possible
presence of free acrylamide.
5. Polyacrylamide gels have a rather small range of separation,
but very high resolving power.
6. Polyacrylamide gels are used extensively for separating and
characterizing mixtures of proteins.
7. In the case of DNA/RNA, polyacrylamide is used for separating
fragments of less than about 500 bp.
 SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis

-separate proteins according to their electrophoretic mobility

-a function of length of polypeptide chain or molecular weight.
-Not on protein charge
-SDS gel electrophoresis of samples having identical charge per unit
mass due to binding of SDS results in fractionation by size.
 SDS anionic detergent gives -ve charge
1. Denatures native proteins to individual polypeptides.
2. When a protein mixture is heated to 100 °C in presence of SDS,
3. the detergent binds polypeptide backbone.
4. It binds to polypeptides in a constant weight ratio of 1.4 g/g of
polypeptide.
5. In this process, the intrinsic charges of polypeptides becomes
negligible when compared to the negative charges contributed
by SDS.
6. Thus polypeptides after treatment becomes a rod like
structure possessing a uniform charge density, that is same
net negative charge per unit length.
7. Mobilities of these proteins becoz molecular weights only.
PCR
 Polymerase chain reaction (PCR)

• The polymerase chain reaction is an extremely

versatile technique for copying DNA.
• PCR allows a single DNA sequence to be copied
(millions of times), or altered in predetermined ways.
• PCR has many variations, like reverse transcription
PCR (RT-PCR) for amplification of RNA, and real-time
PCR (qReal Time -PCR) which allow for quantitative
measurement of DNA or RNA molecules.
 PCR: Polymerase Chain Reaction
(Technique of xeroxing DNA)
A technique that allows amplification of a specific region/fragment of
DNA and generates thousands to millions of copies of it.
Components of PCR reaction:
• DNA Template that contains the region/fragment to be amplified.
• Two Primers (synthetic oligonucleotides complementary to the 5’ and 3’ ends of
the DNA fragment
• DNA Polymerase
• Four dNTPs (Deoxyribonucleotides triphosphates dATP, dGTP, dCTP and dTTP):

PCR machine: Thermocycler which is used for heating and cooling the reaction sample
at different temperatures.
 Three processing steps of PCR
(1) Denaturation
(2) Annealing
(3) Elongation
(4) Cycle repeated 25 to 30 times

1. Denaturation: Sample heated at 95–98 °C and the two strands of DNA

gets separated.

2. Annealing: Sample temperature is reduced to ~60°C and this leads to

binding of primers to their complementary sequence in the DNA
template.

3. Elongation: Sample temperature is raised to ~72°C and the DNA

polymerase extends the primers by adding four dNTPs complementary
to the DNA template and this results in the synthesis of new DNA.

4. This cycle is repeated number of times (30-40 cycles) using PCR

machine and leads to synthesis of DNA.
95°C

60°C

72°C
PCR: Polymerase Chain Reaction
 PRIMER
• A primer is a strand of nucleic acid that serves as a starting point
for DNA synthesis.
• These primers are usually short, chemically synthesized
oligonucleotides, with a length of about 18 - 30 bases. They are
hybridized to a target DNA, which is then copied by the
polymerase.
• minimum primer length used in most applications is 18
nucleotides.
• Replication starts at the 3'-end of the primer, and copies the
opposite strand.
 General concepts for PRIMER DESIGNING
1. Length of about 18 - 30 bases
2. Ideal GC content is 40-60% (play around with length)
3. Calculated melting temperatures (Tm) should be from 42-65°C
4. Pick primers that don't form hairpins. These can be excluded using a
computer program (IDT oligo analyzer program). A GC-rich hairpin is more
stable (and worse) than an AT-rich one. For example
GCTACGGGGCCCCTGTG would likely fold in the center forming four G-C
bonds and fail to hybridize with template DNA.
5. Avoid primer-primer hybridization. Hairpins are an example of intraprimer
hybridization. Another example is when reverse and forward primers bind to
each other.
6. Melting temperatures of the two primers should be close. Primers should be
hybridized under fairly stringent conditions at (Tm - 10°C) <= Ta <= (Tm - 5°C).
If Tm is 60C then annealing temp range one should try 50C-55c).

Forensic investigations use standard primers that hybridize to highly conserved

DNA regions but which, taken as a pair, span highly variable regions.

1. Bioinfo tools for designing primer: Primer3

2. IDT oligoanalyzer (analysis of designed primer)
 Nucleic acid thermodynamics is the study of
how temperature affects the nucleic acid structure of double-
stranded DNA (dsDNA).

The melting temperature (Tm) is defined as the temperature at

which half of the DNA strands are single-stranded (ssDNA) state.

A=T Less energy needed to break AT

G C More energy needed to break GC

Thermophilic organisms can have %GC = 80% which is why their DNA doesn't
denature at temperatures greater than 100°C.
 DNA has a maximum in optical absorbance at 260 nm.
ssDNA has a greater absorbance due to lower degree of order.
The bases become unstacked and can thus absorb more light.
The two strands of DNA are bound together mainly by the stacking interactions,
hydrogen bonds and hydrophobic effect between the complementary bases.
 DNA Polymerase used in PCR
• Taq polymerase is a thermostable DNA polymerase named after
the thermophilic bacterium Thermus aquaticus from which it was originally
isolated
• T. aquaticus lives in hot springs and hydrothermal vents,
• Taq's optimum temperature for activity is ~72°C,
• Extension rate ~ 50 nt/sec
• One of Taq's drawbacks is its relatively low replication fidelity. Because it lacks
 exonuclease proofreading activity
• It has an error rate measured at about 1 in 9,000 nucleotides
• Eg
Need to amplify a gene coding 20kDa protein
20000Da = 181 residues = 543 nt long DNA to be amplified
Extension time at 72°C will be for ~11 sec
 DNA Polymerase used in PCR
• Pfu  polymerase is a thermostable DNA polymerase named after
hyperthermophilic archaeon Pyrococcus furiosus, from which it was originally isolated
• Pfu's optimum temperature for activity is ~72°C,
• Extension rate 1000 nt/min
• It possesses 3' to 5' exonuclease proofreading activity,
• Means it corrects nucleotide-misincorporation errors. This means that Pfu DNA
polymerase-generated PCR fragments will have fewer errors/mutations than Taq-
generated PCR . 
• An error rate of 1 in 1.3 million base pairs
• Eg
Need to amplify a gene coding 20kDa protein
20000Da = 181 residues = 543 nt long DNA to be amplified
Extension time at 72°C will be for ~30 sec
 Quantifying the DNA

• Nucleic acids have a peak absorbance in the ultraviolet range at about 260
nm

•       1 A260 O.D. unit for dsDNA = 50 µg/ml

•       1 A260 O.D. unit for ssDNA = 33 µg/ml

•       1 A260 O.D. unit for RNA = 40 µg/ml

 Molecular Weight DNA/RNA
The approx.
•dsDNA basepair (bp) weight is 650 Dalton,
•ssDNA is ~330 Da/base,
•RNA is ~340 Dalton/base.

If only DNA length is given, the molecular weight is calculated as:

MW = DNA Length (bp) × DNA/RNA base weight

 Purity of DNA
• 260/280 ratio = 1.8 pure DNA
• 260/280 ratio = 12 pure RNA
• If less means contamination of proteins or
something else

• Purity of Protein
% protein % nucleic acid 260:280 ratio

100 0 0.57
 Electroblotting

 A method or a technique in biotechnology to

transfer proteins or nucleic acids onto a membrane
(PVDF/PolyVinyliDene Fluoride  or nitrocellulose) after gel
electrophoresis.

The protein or nucleic acid can then be further analyzed

using probes which specifically bind to protein or nucleic
acid.

Probes for
Proteins = antibodies, 
Nucleic acid = complementary oligo-nucleotides
 Electroblotting

Protein (SDS-PAGE)/DNA/RNA all are negatively charged and move

towards anode (+).

Therefore gel containing sample is on cathode side and membrane is

on anode side
 Types of Blotting

The transfer of separated proteins from

polyacrylamide gel onto a membrane such as
nitrocellulose (NC) is called WESTERN
BLOTTING.

The transfer of DNA from agarose gel onto

NC is called SOUTHERN BLOTTING

The transfer of RNA from agarose gel onto

NC is called NORTHERN BLOTTING
Detection of DNA in sample
‘Southern’ hybridization named after
Sir Edwin Southern
Developed in 1975
Spawned naming of related techniques:

Northern blot
(RNA)

Western blot Eastern blot

(Protein) (PMT: Post translational
modifications such as lipids,
phosphomoieties and
glycoconjugates)
Southern blot
(DNA)
Immobilize DNA onto a permanent substrate

Identify DNA sequence (gene) of interest using a probe

(labeled complementary oligonucleotides)
Arabidopsis thaliana 2 copies of gene X
 extract

DNA

Capsella rubella ? copies of gene X

EcoR I EcoR I EcoR I EcoR I
_ +
_ +
 Immobilize/electroblot DNA onto a permanent substrate

‘Membrane’
 paper-like matrix
 nylon or nitrocellulose
 usually has a slight positive charge
• Eliminate hydrogen bonds with sodium
hydroxide (NaOH)
• Soak gel in alkali (0.4 M NaOH) ~30 mins

A C T T G A

T G A A C T
• Two methods for transferring DNA to a
membrane
– capillary
– electrophoretic
Immobilize DNA onto a permanent substrate

Identify DNA sequence (gene) of interest using labeled

oligonucleiotide probe
A probe is a small (25-2000 bp) length of oligonucleotide
 Complementary to the sequence (gene) of interest
 Labeled for subsequent detection procedures
Arabidopsis thaliana 2 copies of gene X
 Gene X
from Arabidopsis

Partial or full-length
probes by PCR
Denature template with heat
Add random primers
Extend random primers with polymerase using PCR
A probe complementary to the sequence (Gene X) of interest!
How do we detect the probe?
 Radioactivity (32P)
How do we detect the probe?
 Digoxigenin (DIG)
 Biotin (Vit B7)

U
Prehybridization buffers
contain ‘blocking reagents’
that occupy available binding
sites on the membrane
CSPD is a chemiluminescent substrate for enzyme alkaline
phosphatase Produces light detected by X-ray film and autoradiography
DIG-labeled probes
emitting minute
amounts of light
(chemiluminescence)

32P-labeled probes
emitting ß-particles
DIG-labeled probes
emitting minute
amounts of light
(chemiluminescence)

32P-labeled probes
emitting ß-particles

Autoradiography film
can detect this
radiation
Gel X-ray film
 How many copies of ‘Gene X’
does Capsella rubella possess?

3
Capsella rubella
Applications of southern blotting
DNA fingerprinting include:
 Paternity and Maternity Testing
 Criminal Identification and Forensics
 Personal Identification
Streptavidin and Avidin
Tetrameric proteins with four specific, high-affinity
binding sites for  biotin.

Biotin, also known as vitamin H/Coenzyme

Biotin labeled oligo probes are also used in

southern blotting .
 Biotin + avidin or streptavidin

Biotin avidin complex

HRP: Horse radish peroxidase is the enzyme conjugated to Streptavidin for detection
 Need to extract intact mRNA from total RNA
 Using oligo dT cellulose/magnetic beads crosslinked to oligo
dT/strepavidin coated magnetic beads capable of binding biotinylated
oligo(dT) which is added to sample and hybridizes to mRNA
 RNA less stable = degraded by RNases = wide prevalence
 Extra precautions taken, DEPC (diethylpyrocarbonate) treated
water/glasswares etc. DEPC inhibits RNase.
 The 2′-hydroxyl group on ribose makes RNA susceptible to alkaline
hydrolysis in solutions of pH greater than nine or so.
 RNA secondary structure = sec st removed by running agarose gels
containing  formaldehyde
 Formaldehyde will hydrogen bond with the bases and denature
RNA, giving single-stranded RNA
 RNA/DNA probes