Gel Electrophoresis
Gel Electrophoresis
• Definition: Separation of DNA fragments according
to size, based on movement through a gel medium
when an electric field is applied.
• When DNA is cut by restriction enzymes, the
result is a mix of pieces of DNA of different
lengths.
• It is useful to be able to separate the pieces ; for
recovering particular pieces of DNA, for forensic
work or for sequencing.
Gel Electrophoresis
• The fragments of DNA can be separated using gel
electrophoresis. Because of its phosphate groups,
DNA is negatively charged at neutral pH.
• When DNA is placed in a semisolid gel and an
electric field is applied, the DNA molecules
migrate toward the positive pole.
• Smaller molecules can migrate more quickly
through the porous gel than larger ones.
• After a fixed time, the separated molecules can
then be stained with a fluorescent dye and
examined under ultraviolet light.
Gel Electrophoresis of DNA
•
•
Separation technique: separates DNA by size and charge
1.Restriction enzymes
– cut DNA I into fragments
•
2. The gel
– Wells made at one end. Small amounts of DNA are placed in the wells
3. The electrical field
gel placed in buffer solution and an electrical filed is set up
4. The fragments move
negatively charged DNA fragments travel toward positive end. The smaller
fragments move faster.
Mixture of DNA
molecules of
different sizes
Longer
molecules
Power
source
Gel
Shorter
molecules
Agarose
• polysaccharide extracted from seaweed. It is
typically used at concentrations of 0.6 to 2%.
The higher the agarose concentration the
"stiffer" the gel. Agarose gels are extremely
easy to prepare: you simply mix agarose
powder with buffer solution, melt it by
heating, and pour the gel.
• non-toxic.
Make up the gel which the DNA will be put
into
Polyacrylamide
• is a cross-linked polymer of acrylamide.
• Polyacrylamide gels are significantly more annoying to
prepare than agarose gels. Because oxygen inhibits the
polymerization process, they must be poured between
glass plates.
• Acrylamide is a potent neurotoxin and should be
handled with care.
Uses
• Polyacrylamide gels have a rather small range of
separation, but very high resolving power.
• polyacrylamide is used for separating fragments of less
than about 500 bp. However, under appropriate
conditions, fragments of DNA differing in length by a
single base pair are easily resolved. In contrast to
agarose, polyacrylamide gels are used extensively for
separating and characterizing mixtures of proteins.
• Agarose is used to separate DNA fragments from about
60 bp upward to 100,000 bp.
Visualization of DNA
• Ethidium bromide, a fluorescent dye used for
staining nucleic acids.
• Teratogen and suspected carcinogen and
should be handled carefully.
• Transilluminator (an ultraviolet light box)
DNA is stained using ethidium bromide
• Most gels have
one lane as a
‘DNA ladder’ DNA fragments
of known size are
used for
comparison
• DNA shows
up as bands
in UV light
Electrophoresis provides information on:
• Size of fragments by comparison to DNA
fragments of known size added to the gel as a
reference.
• Presence of specific sequences by using a
complementary labeled single-stranded DNA
probe
• DNA is denatured while in the gel, then
transferred to a nylon filter to make a “blot.”
• Also the specific fragment can be cut out as a
lump of gel and extracted by diffusion into a
small volume of water to be sequenced or for
any further study.
Southern Blots
• Way to detect specific DNA segments
• Transfers DNA from gel to nitrocellulose filter
• Identify specific DNA segment with
radioactive, single-stranded DNA probe
– Northern blots: RNA
– Western blots: protein
Southern blotting - Procedure
• Technique developed by Ed Southern used for variety
of purposes
• Procedure:
– 1. DNA is digested with restriction enzymes and separated
by agarose gel electrophoresis
– 2. Gel is treated with NaOH to denature DNA ss DNA
– 3. DNA is transferred from gel to a DNA-binding filter (e.g.
nitrocellulose or nylon membrane) using capillary action
• Gel sits on a sponge wick. Paper towels absorb rising
buffer.
• Buffer passes through the membrane but not the DNA.
• DNA binds to membrane
– 4. DNA is “fixed” by heating membrane at 80oC
– 5. The membrane is incubated with ss-nucleic
acid probe binds to DNA is complementary.
Remainder washed off.
– 6. Autoradiography for detection of DNA
fragment of interest .
Southern Blots
• Radioactive probes are used .
Analysis of DNA Polymorphisms in Genomes
Polymrphisms are inherited differences found
among the individuals in a population.
There are three major classes of DNA
polymorphisms:
a.Single nucleotide polymorphisms (SNPs)
b.Short tandem repeats (STRs)
c. Variable number tandem repeats (VNTRs)
Single nucleotide polymorphisms (SNPs, “snips”) are base-pair differences
between individuals. SNPs account for 90–95% of sequence variation, and
arise by spontaneous mutation.
a. Most SNPs occur in noncoding regions of the genome. Noncoding SNPs can affect
gene function if they are in gene regulatory regions such as promoters.
b. SNPs in coding regions (cSNPs) cause missense mutations (about half) or silent
mutations (the other half). The average gene has about four SNPs.
c. A few SNPs create or abolish restriction sites, resulting in restriction fragment
length polymorphisms (RFLPs). RFLPs are detected by:
i. Southern hybridization:
(1) Isolates genomic DNA and digest with a restriction enzyme.
(2) Electrophoreses and transfers DNA to a membrane filter.
(3) Probes with labeled DNA from the polymorphism region.
(4) Monozygotes show one band, heterozygotes two.
ii. PCR amplification :
(1) Isolates genomic DNA and amplifies sequence of interest with specific
primers.
(2) Digests amplified DNA with appropriate restriction enzyme.
(3) Analyzes fragments produced with agarose gel electrophoresis.
Fig.
Southern blot analysis method for studying SNP that
affect restriction sites
PCR method for studying SNP that
affect restriction sites
Short tandem repeats (STRs), or microsatellite sequences, contain very short (2–6bp)
tandem repeats, and are highly polymorphic.
a.
b.
c.
d.
Examples are the dinucleotide repeat (GT)n and the trinucleotide repeat (CAG)n.
STRs are distributed widely in the human genome, with thousands of sites currently known.
Many are polymorphic and are used for genetic mapping and forensics.
STRs are usually typed by PCR with primers flanking the sequence (Figure 9.6).
i. A population may have many different allele lengths for STRs.
ii. An individual may be either homozygous or heterozygous for a particular STR.
Variable number of tandem repeats (VNTRs), also called minisatellites, are longer than
STRs (7 or more bp).
a. There are far fewer VNTR loci in the human genome than STR loci.
b. To detect VNTR polymorphisms, PCR is not generally useful, and instead, restriction digestion
and Southern blotting are used.
i. DNA is digested with a restriction enzyme that cuts flanking the VNTR.
ii. Fragments are electrophoresed, and blotted to a filter.
iii. The blot is probed with the VNTR repeating sequence.
(1)Some VNTR sequences are in only one genomic locus, corresponding to a monolocus
probe.
(2)Other VNTR sequences map to a number of genomic loci, corresponding to a multilocus
probe.
Using PCR to determine which STR (microsatellite) alleles
DNA Fingerprinting with Short Tandem Repeats
DNA Fingerprinting
• Very specific form of RFLP analysis
• Used to:
– identify hereditary relationships
– study inheritance of patterns of diseases
– study human evolution
– identify criminals or victims of disaster
DNA Fingerprinting
• Uses VNTRs or STR
– variable number of tandem repeats
– All humans have the same type of repeats but there is
tremendous variation in the number of repeats that each
has :
– Short, repeated sequences (~ 4 bases)
• GGACGGACGGACGGAC
CC TGCCTGCC TGCCTG
– Non-coding, highly variable between individuals
– STRs are recognizable if they lie between two restriction
sites.
– Several different STRs can be used to determine the unique
pattern for an individual.
• In the non-coding regions of the genome, sequences of DNA are frequently
repeated giving rise to variable number of tandem repeats.
• The number of repeats varies between different people and can be used to
produce their genetic fingerprint.
• In example shown above, person A has only 4 repeats whilst person B has 7.
• When their DNA is cut with the restriction enzyme Eco RI, which cuts the
DNA at either end of the repeated sequence (in this example), the DNA
fragment produced by B is nearly twice as big as the piece from A, as shown
when the DNA is run on a gel.
• The lane marked M contains marker of DNA that help to determine the sizes.
• If whole genomic DNA of individual is analysed in this way, a 'fingerprint'
comprising DNA fragments of different sizes, unique to every individual,
emerges
DNA Fingerprinting
• STEPS:
1. Sample DNA cut with restriction enzymes
2. Fragments separated by size using gel electrophoresis
3. Fragments with highly variable regions are detected
with DNA probe, revealing DNA bands of various sizes
4. The pattern of bands produced is the DNA fingerprint,
which is distinguished statistically form other individuals
DNA fingerprinting requires at least 1 μg of DNA
(amount in about 100,000 human cells). This is not
always available, so amplification by PCR is used.
Practical Applications of DNA
Fingerprinting
• 1.Paternity and Maternity
• person inherits his or her STRs and VNTRs
from his or her parents
• Parent-child TRs pattern analysis has been
used to solve standard father-identification
cases
DNA Fingerprinting in a Paternity Case
Paternity case would proceed as follows :
a. DNA samples (typically from blood) are obtained from mother, baby
and putative father.
b. DNA is cut with a particular restriction enzyme, electrophoresed and
transferred to a membrane filter by Southern blotting.
c. Hybridization is performed with a labeled monolocus STR or VNTR
probe, and the banding pattern is analyzed.
d. Baby and mother are expected to share one allele, while baby and
father share the other allele.
e. If the man and baby do not share a common allele, DNA typing has
proved he is not the father. If they do share an allele, paternity is
possible, but not proven, since other men also carry the allele at some
frequency that can be calculated.
f. Often five or more different polymorphisms are characterized. If all
match with the putative father, the combined probabilities calculated
for the array of polymorphisms can be convincing evidence in court.
Procedure for DNN Fingerprinting as used for a paternity case
DNA Fingerprinting - Paternity Test
mother
child
Who is the possible father ?
Mr. A
Mr. B
Mr. C
DNA Fingerprinting - Paternity Test
mother
child
Who is the possible father ?
Mr. A
Mr. B
Mr. C
2. Criminal Identification and Forensics
• DNA isolated from blood, hair, skin cells, or other
genetic evidence left at the scene of a crime can be
compared
• FBI and police labs around
the U.S. have begun to use
DNA fingerprints to link suspects
to biological evidence –
blood or semen stains, hair,
or items of clothing
• In the example shown, DNA collected at the scene of a crime is compared
with DNA samples collected from 4 possible suspects. The DNA has been cut
up into smaller pieces which are separated on a gel. The fragments from
suspect 3 match those left at the scene of the crime .
•DNA fingerprinting is used in forensics.It is more often used to prove innocence than guilt.
• Only a small portion of the genome is examined; there is the possibility that two people
could have the same sequence.
3. To analyze historical events.
The skeletal remains of Russian Tsar
Nicholas II and his family were identified
from DNA in bone fragments.
DNA also showed relationships with living
descendents of the Tsar.
DNA Fingerprinting the Russian Royal Family
Considerations when evaluating DNA
evidence
• In the early days of the use of
genetic fingerprinting as criminal
evidence, given a match that had a
1 in 5 million probability of occurring
by chance the lawyer would argue
that this meant that in a country
of say 60 million people there were 12 people
who would also match the profile.
• The final DNA fingerprint is built by using
several restriction enzymes and several
probes (5-10 or more) simultaneously.
Examples of DNA Molecular Testing
Testing by restriction fragment length polymorphism (RFLP) analysis
detects loss or addition of a restriction site in the region of a gene.
RFLPs are associated with many genetic disorders. Sickle-cell anemia
is an example:
a.
b.
c.
A single base-pair change in the β-globin gene results in abnormal
hemoglobin, Hb-S, rather than the normal Hb-A. Hb-S molecules
cause sickling of red blood cells.
The Hb-S mutation is an AT-to-TA base pair change in the 6th codon
of β-globin, resulting in a valine rather than a glutamic acid, and
also eliminating a DdeI restriction enzyme site .
In the normal β-globin (Hb-A) gene there are three DdeI sites,
while the sickling form, Hb-S, has only two DdeI sites. This
difference can be detected using Southern hybridization of
genomic DNA with a β-globin gene probe .
Normal Hb-A sequences and the mutant Hb-S sequences
Detection of sickle-cell gene by the DdeI restriction fragment
length polymorphism
RFLPs associated with genetic disorders may also result from
changes in flanking sequences. PKU is an example:
a. PKU results from defective phenylalanine hydroxylase enzyme.
b. Genomic DNA digested with HpaI, Southern blotted and probed
with cDNA probe from phenylalanine hydroxylase mRNA shows
different restriction fragments for PKU and normal individuals.
c. The RFLP results from DNA sequences located 3’ to the gene
that usually segregate with it. Recombination events that occur
between the site of the RFLP and the gene mutation can
complicate this test.
Examples of tests for specific mutations using blood from
newborns include:
a. Phenylketonuria (PKU).
b. Sickle-cell anemia.
c. Tay-Sachs disease.
Carrier detection tests using blood samples are available
for many genetic diseases, including:
a. Tay-Sach disease.
b. Duchenne muscular dystrophy (a disease of progressive muscle
atrophy and disfunction).
c. Cystic fibrosis.
Pedigree analysis
43
Purposes of Human Genetic Testing
1. Human genetic testing serves three main purposes:
a. Prenatal diagnosis.
b. Newborn screening.
c. Carrier (heterozygote) detection.
2. Prenatal diagnosis uses amniocentesis or chorionic villus sampling
to assess risk to the fetus of a genetic disorder by analyzing for a
specific mutation, or biochemical or chromosomal abnormalities.
a. If both parents are carriers (heterozygotes) for the mutant allele,
the probability is 1⁄4 that the fetus is an affected homozygote,
1/2 that it is a carrier, and 1⁄4 that it is homozygous for the
normal allele. Genetic testing can determine the result of a
particular conception.
b. Genetic testing may be used during in vitro fertilization to
eliminate before implantation embryos with mutated genes that
could result in serious disease.
Polymerase chain reaction
• PCR is the in vitro enzymatic synthesis and
amplification of specific DNA sequences.
• Can amplify one molecule of DNA into
billions of copies in a few hours.
• PCR was previously studied.