Basic Principles and Applications
of Electrophoresis
Stephen K.W. Tsui
Department of Biochemistry
Order Form for Electrophoresis
Kits and Reagents
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Theory of Electrophoresis
The movement of a charged molecule subjected to an
electric field is represented by the following equation:
V =
Eq
f
V:
the velocity of the molecule
E:
the electric field in volts/cm
q:
the net charge on the molecule
f:
frictional coefficient, which depend on the
mass and shape of the molecule
Applications of Gel Electrophoresis
• Southern blot is produced when
DNA on a nitrocellulose blot is
hybridized with a DNA probe.
• Northern blots are generated
when RNA is hybridized with a
complementary DNA probe
produced by the reverse
transcription of messenger RNA.
• A slightly different but related
technique, known as a Western
blot, involves separating proteins
by gel electrophoresis and
probing with labeled antibodies
for specific proteins.
Blotting Techniques
Southern Blot
Northern
Blot
Western
Blot
Macromolecules
on the blot
DNA
RNA
Protein
Probe
Labeled DNA
Labeled DNA
Labeled
antibodies
Source of labels
Radioactively or
fluorescent
labeled
deoxynucleotide
Radioactively or
fluorescent
labeled
deoxynucleotide
Radioactively or
fluorescent
labeled amino
acids
Tissue distribution of Messenger
RNA Revealed by Northern Blot
1 2 3 4 5 6 7 8
9 10 11 12 13 14 15 16
1: heart
2: brain
3: placenta
4: lung
5: liver
6: skeletal
muscle
7: kidney
8: pancreas
9 : spleen
10: thymus
11: prostate
12: testis
13: ovary
14: small intestine
15: colon
16: leukocyte
A Protein can be Specifically Recognized
by an Antibody in a Western Blot
M 1 2 3 4
M 1 2 3 4
Coomassie Blue Dye
Stained protein gel
Western blot
Gel Electrophoresis of DNA
Wells for sample
loading
Cathode (-)
Direction for DNA
migration
Anode (+)
Agarose slab gel submerged
in buffer
Agarose is a polysaccharide derived from seaweed, which
forms a solid gel when dissolved in aqueous solution. When an
electric field is applied to an agarose gel in the presence of a
buffer solution which will conduct electricity, DNA fragments
move through the gel towards the positive electrode (DNA is
highly negatively charged) at a rate which is dependent on its
size and shape.
Gel Electrophoresis of DNA
For linear DNA molecules, they have uniform
shape and charge to mass ratio. The
electrophoretic mobility of the DNA molecule
is influenced primarily by the molecular size:
The larger molecules are retarded by the
molecular sieving effect of the gel, and the
small molecules have greater mobility.
Gel Electrophoresis of DNA
• The DNA can be stained by the inclusion of ethidium
bromide in the gel, or by soaking the gel in a solution
of ethidium bromide after electrophoresis. The DNA
shows up as an orange band on illumination by UV
light. Alternatively, methylene blue can be used to
stain DNA.
• Gels composed of polyacrylamide can separate DNA
molecules that differ in length by only one nucleotide
and are used to determine the base sequence of DNA.
Agarose gels are used to separate DNA fragments that
have larger size differences.
Procedures of DNA Fingerprinting
Procedures of DNA Fingerprinting
• In order to detect specific sequences, DNA is usually
transferred to a solid support, such as a sheet of
nitrocellulose or nylon paper.
• The paper is treated with an alkaline solution to
denature DNA, that is, separate the two strands of
each double helix.
• The single-stranded DNA can be hybridized with a
probe, and the regions on the nitrocellulose blot
containing DNA that base-pairs with the probe can be
identified.
DNA Polymorphisms
• Polymorphisms are variations in DNA sequences.
There may be millions of different polymorphisms in
the human DNA.
• Polymorphisms in the human DNA serve as the basis
for the diagnosis of diseases and the identity of
individuals.
Detection of Polymorphism
Restriction Fragment Length Polymorphisms
• Occasionally, a point mutation occurs in a recognition
site for a restriction enzyme. The enzyme, therefore,
can cut at other recognition sites but not at the site of
the mutation. Consequently, the restriction fragment
produced by the enzyme is larger for a person with the
mutation than for a normal person.
• Mutations can also create restriction sites that are not
present in the normal gene. In this case, restriction
fragments will be smaller for the person with the
mutation than for the normal individual. These
variations in the length of restriction fragments are
known as restriction fragment length polymorphisms
(RFLPs).
Application of DNA Fingerprinting
Mutation Detection
Highly Variable Regions
• Human DNA contains many sequences that
are repeated in tandem a variable number of
times at certain loci in the genome. These
regions are called hypervariable regions
because they contain a variable number of
tandem repeats (VNTR).
Detection of Highly Variable Regions
Digestion with restriction enzymes that recognize sites which
flank the VNTR region produces fragments containing these loci,
which differ in size from one individual to another, depending on
the number of repeats that are present. Probes used to identify
these restriction fragments bind to or near the sequence that is
repeated.
Application of DNA Fingerprinting
Forensic Analysis
This restriction fragment technique has been called "DNA
fingerprinting" and is gaining widespread use in forensic analysis.
Family relationships can be determined by this method, and it can be
used to convict suspects in criminal cases. Individuals who are
closely related genetically will have restriction fragment pattern that
are more similar than those who are more distantly related.
Other Applications of DNA Fingerprinting
• Parentage test
• Endangered species or Chinese herbs
identification
Animation 1: Southern Blotting
http://www.dnalc.org/resources/BiologyAnimatio
nLibrary.htm
Animation 2: DNA Detective
http://www.dnalc.org/resources/BiologyAnimatio
nLibrary.htm
Online Courses: DNA from the Beginning
http://www.dnaftb.org/dnaftb/
Download Illustrations:
Human Molecular Genetics
http://www.bios.co.uk/illustrations.asp
Good Website: Gel Electrophoresis
http://dlab.reed.edu/projects/vgm/vgm/VGMPr
ojectFolder/VGM/RED/RED.ISG/gel.html
Workshop
Agarose Gel Electrophoresis
Department of Biochemistry
(2001-2002)
Properties of DNADouble helix
Building block(dA, dC, dG and dT)
negatively charged at neutral pH
AT and GC complementary pairing
Restriction enzymes - enzymes isolated from
bacteria that cut DNA at specific
sites(restriction sites)
EcoRI - 5'- G A A T T C -3‘
3'- C T T A A G -5'
Baterial plasmid DNA
Plasmids are molecules of DNA that are found in
bacteria separate from the bacterial chromosome.
They:
are small (a few thousand base pairs)
usually carry only one or a few genes
are circular
have a single origin of replication
Plasmid DNA for digestion
– pBluescript II SK+
BglI 472
BglI 2166
+
EcoRI 701
Agarose
A linear polymer extracted from seaweed
Migration of DNA in agarose dependent
on four factors
- molecular size of the DNA
- agarose concentration
- conformation of the DNA
- applied current
Cathode(-)
wells
DNA
fragments of
different sizes
1.5% agarose
gel stained with
methylene blue
Anode(+)
Preparation of
plasmid DNA
http://dlab.reed.edu/projects/vgm/vgm/V
GMProjectFolder/VGM/RED/RED.ISG/
gel.html
Restriction
enzyme
digestion
http://dlab.reed.edu/projects/v
gm/vgm/VGMProjectFolder/
VGM/RED/RED.ISG/gel.htm
l
» Agarose gel casting
» DNA sample loading
» electrophoresis
Methylene
blue staining
Agarose gel electrophoresis unit
Electrophoresis tank
Plugs and wire
Gel casting unit
and comb
Agarose gel casting unit
Step 4
Comb
Tape
Gel casting unit
Seal both ends of the
gel casting unit with
tape
Preparation of 1.5% agarose gel
Step 5
http://dlab.reed.edu/projects/vgm/vgm/VGMPro
jectFolder/VGM/RED/RED.ISG/gel.html
Step 10
Sample loading,
wash syringe with 1X
TBE buffer between
successive loading
http://dlab.reed.edu/projects/vgm/vgm/V
GMProjectFolder/VGM/RED/RED.ISG/
gel.html
Electrophoresis(5V/cm)
Wells
Tracking dye
Xylene cyanol
FF
Bromophenol
blue
Methylene blue staining
» to visualize the DNA fragments, stain
agarose gel overnight with 1X methylene blue
staining solution
» safe alternative for DNA staining
» easy available
» non-carcinogenic
DNA
fragments
DNA
fragments of
different sizes
A B
DNA
fragments of
known sizes
C D M
Base pairs
distance
migrated(mm)
1808
1634
23
25
656
38
316
47
Calibration curve for DNA size
determination
http://www.pangloss.com/seidel/Protocols/webmap.html
Size determination of the candidate DNA
fragments
http://www.pangloss.com/seidel/Protocols/webmap.html
Workshop
DNA Fingerprinting
&
Agarose Gel Electrophoresis
Department of Biochemistry
CUHK
(TDC2003)
Animal cell
nucleus
Human genome ~ 3 billion base pairs.
5 % of the genome are protein coding sequence
(30,000 genes).
95% non-coding DNA.
20-30% are repetitive.
Repetitive DNA
Tandemly repeated sequences
(~10% of genome)
Satellite DNA
Minisatellites / VNTRs
Interspersed elements
(~15-20% of the genome)
SINES eg. Alu
(3-6%)
LINES eg L1
(1-2%)
Rich source of genetic variation
VNTRs : Variable number tandem repeats
(J.C.S. Fowler et al.)
Identical twins
You and me
0.1 % vary
person to
person
Each of us have unique DNA fingerprint /
personal barcode.
Two kinds of DNA polymorphic regions (regions of different)
Sequence polymorphism
A, T
Length polymorphism
Simple variations in the physical
length of the DNA(eg. VNTRs)
…ACGTAGCAGCAGCG…
…TGCATCGTCGTCGC…
T, A
Simple substitution of one or two bases in the gene
DNA fingerprint/Length polymorphism in a
single locus
M P
M P
M P
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
AT
Person 1
Homozygous
Person 2
Heterozygous
Person 3
Heterozygous
AT
VNTR repeating unit
M
P
Maternal chromosome copy
Paternal chromosome copy
Variable number tandem repeats (VNTRs)
M P
VNTRs are not distributed evenly across human population.
Each of allele occurs at a certain frequency in a population.
AT
AT
AT
Each locus usually has approximately 30 different alleles.
Frequency of allele A at one locus = 0.1 (10%)
Frequency of allele B at second locus = 0.05 (5%)
Frequency of the two alleles of the loci occur together = 0.1 X 0.05
(DNA profile frequency)
= 0.005
or 1 in 200
AT
AT
AT
AT
AT
AT
AT
AT
AT
Locus X
Locus Y
Chromosome A
Chromosome B
Locus Z
X
Y
Z
Father
10, 14
18, 24
8, 9
Mother
12, 20
22, 22
8, 15
Son
10, 20
22, 24
8, 8
Daughter
12, 14
22, 22
26, 9
DNA profile frequency for 3 loci: ~1 in 3 million
Application of DNA fingerprinting
Paternity and maternity test.
Criminal identification and forensics.
Personal identification.
Source of human DNA for fingerprinting
Whole blood
Buccal epithilial cells
Hair follicles
Semen
Polymerase chain reaction
Double stranded target DNA
Step 1 : Denaturation
Two DNA targets available for PCR
95oC
DNA denatured
PCR
amplification
Cycle
(35 – 40 cycles)
Step 3 : DNA Extension
Step 2 : Primer Annealing
~55oC
Primers bind to target DNA
72oC
Double stranded DNA duplicated
Amount of amplified DNA = 2n x C
where n = number of PCR cycles; and
C = the initial number of copies of DNA template present in the tube.
So, you will get 1,048,576 copies of DNA after 20 cycles of PCR reaction even you start with
only one copy of DNA template initially
Maternal tandem
repeat fragment
Ethidium
bromide stained
agarose gel
Paternal tandem
repeat fragment
Home made gel electrophoresis kit
10 X TBE
concentrate
Electrical wire
1ml syringe
filtted with tip
Gel casting unit
Electrophoresis unit
Agarose
A linear polymer extracted from seaweed
Migration of DNA in agarose dependent
on four factors
- molecular size of the DNA
- agarose concentration
- conformation of the DNA
- applied current
Agarose gel casting unit
Comb
Tape
Gel casting unit
Seal both ends of the
gel casting unit with
tape
Preparation of 1.5% agarose gel
Sample loading
Wells
Electrophoresis(5V/cm)
DNA is negatively charg
Power supply
Nine 9V batteries
connected in series
Three 24V adaptor
connected in series
Cathode (-)
Wells
Tracking dye
Xylene cyanol
FF
Bromophenol
blue
1cm
Anode (+)
Methylene blue staining
» to visualize the DNA fragments, stain
agarose gel overnight with 1X methylene blue
staining solution and destain in distilled water
for 3 – 4 hours
» non-toxic
» easy available
» non-carcinogenic
Results
MK M F D1 D2 S1 S2
2100 bp
1700 bp
1100 bp
Lane MK:
Lane M:
Lane F:
Lane D1:
Lane D2:
Lane S1:
Lane S2:
850 bp
600 bp
250 bp
(Biologic child / adopted child / stepchild)
DNA marker
Mother
Father
Daughter 1
Daughter 2
Son 1
Son 2
Calibration curve for DNA size
determination
Size determination of the candidate DNA
fragments
online size determination
THE END