Basic Principles and Applications of Electrophoresis

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Basic Principles and Applications of
Electrophoresis
Stephen K.W. Tsui Department of Biochemistry
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Order Form for Electrophoresis Kits and Reagents
Please click this sentence to download the form
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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 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
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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.

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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
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Tissue distribution of Messenger RNA Revealed by
Northern Blot
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
1 2 3 4 5 6 7 8
9 10 11 12 13 14 15 16
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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
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Gel Electrophoresis of DNA
 
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.
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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.
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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.

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Procedures of DNA Fingerprinting
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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.

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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.

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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).

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Application of DNA Fingerprinting Mutation
Detection
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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).

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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.
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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.
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Other Applications of DNA Fingerprinting

• Parentage test
• Endangered species or Chinese herbs identification

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Animation 1 Southern Blotting
http//www.dnalc.org/resources/BiologyAnimationLib
rary.htm
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Animation 2 DNA Detective
http//www.dnalc.org/resources/BiologyAnimationLib
rary.htm
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Online Courses DNA from the Beginning
http//www.dnaftb.org/dnaftb/
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Download Illustrations Human Molecular Genetics
http//www.bios.co.uk/illustrations.asp
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Good Website Gel Electrophoresis
http//dlab.reed.edu/projects/vgm/vgm/VGMProjectFo
lder/VGM/RED/RED.ISG/gel.html
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The End
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Workshop Agarose Gel Electrophoresis Department
of Biochemistry (2001-2002)
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Properties of DNA- Double helixBuilding
block(dA, dC, dG and dT)negatively charged at
neutral pHAT and GC complementary pairing
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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
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Plasmid DNA for digestion pBluescript II SK

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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

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Cathode(-)
wells
DNA fragments of different sizes
1.5 agarosegel stained with methylene blue
Anode()
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Preparation of plasmid DNA
http//dlab.reed.edu/projects/vgm/vgm/VGMProjectFo
lder/VGM/RED/RED.ISG/gel.html
http//dlab.reed.edu/projects/vgm/vgm/VGMProjectFo
lder/VGM/RED/RED.ISG/gel.html
Restriction enzyme digestion

• Agarose gel casting
• DNA sample loading
• electrophoresis

Methylene blue staining
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Agarose gel electrophoresis unit
Electrophoresis tank
Plugs and wire
Gel casting unit and comb
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Agarose gel casting unit
Step 4
Comb
Tape
Gel casting unit
Seal both ends of the gel casting unit with tape
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Preparation of 1.5 agarose gel
Step 5
http//dlab.reed.edu/projects/vgm/vgm/VGMProjectFo
lder/VGM/RED/RED.ISG/gel.html
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Step 10
Sample loading, wash syringe with 1X TBE buffer
between successive loading
http//dlab.reed.edu/projects/vgm/vgm/VGMProjectFo
lder/VGM/RED/RED.ISG/gel.html
Electrophoresis(5V/cm)
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Wells
Tracking dye
Xylene cyanol FF
Bromophenol blue
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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

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DNA fragments of different sizes
DNA fragments of known sizes
A B C D M
Base pairs distance
migrated(mm)
1808 23 1634 25
656 38
316 47
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Calibration curve for DNA size determination
http//www.pangloss.com/seidel/Protocols/webmap.ht
ml
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Size determination of the candidate DNA fragments
http//www.pangloss.com/seidel/Protocols/webmap.ht
ml
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Workshop DNA Fingerprinting Agarose Gel
Electrophoresis Department of BiochemistryCUHK(
TDC2003)
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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.

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Repetitive DNA
Interspersed elements(15-20 of the genome)
Tandemly repeated sequences (10 of genome)
Satellite DNA
Minisatellites / VNTRs
SINES eg. Alu (3-6)
LINES eg L1(1-2)
VNTRs Variable number tandem repeats
(J.C.S. Fowler et al.)
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Identical twins
You and me
0.1 vary person to person

• Each of us have unique DNA fingerprint /
  personal barcode.

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Two kinds of DNA polymorphic regions (regions of
different)
Sequence polymorphism
Length polymorphism
Simple variations in the physical length of the
DNA(eg. VNTRs)
ACGTAGCAGCAGCG
TGCATCGTCGTCGC
Simple substitution of one or two bases in the
gene
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DNA fingerprint/Length polymorphism in a single
locus
Person 1Homozygous
Person 2Heterozygous
Person 3Heterozygous
VNTR repeating unit
AT
M Maternal chromosome copy
P Paternal chromosome copy
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Variable number tandem repeats (VNTRs)

• VNTRs are not distributed evenly across human
  population.
• Each of allele occurs at a certain frequency in
  a population.
• 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

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Locus X Locus Y
Chromosome A
Chromosome B
Locus Z
DNA profile frequency for 3 loci 1 in 3 million
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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

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Polymerase chain reaction
                                 
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
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Maternal tandem repeat fragment
Ethidium bromide stained agarose gel
Paternal tandem repeat fragment
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Home made gel electrophoresis kit
10 X TBE concentrate
Electrical wire
1ml syringe filtted with tip
Gel casting unit
Electrophoresis unit
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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

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Agarose gel casting unit
Comb
Tape
Gel casting unit
Seal both ends of the gel casting unit with tape
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Preparation of 1.5 agarose gel
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Sample loading
Wells
Electrophoresis(5V/cm)
DNA is negatively charged
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Power supply
Nine 9V batteries connected in series
Three 24V adaptor connected in series
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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

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Results

MK M F D1 D2 S1 S2


(Biologic child / adopted child / stepchild)
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Calibration curve for DNA size determination
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Size determination of the candidate DNA fragments
online size determination
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THE END