Comparing DNA

 Organisms that have similar phenotypes

have similar DNA sequences.
 DNA between two organisms can be
compared in a couple of different ways:
– DNA fingerprinting
– DNA sequencing

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DNA Fingerprinting:
 is the process of analyzing genetic material
from an individual to determine identity,
susceptibility to disease and other important
information;
 has increased in popularity in the recent
years in the fields of criminology,
biomedicine, biotechnology and family law;
 proves how much related these people are,
and if two people having the same surname
are related;
 can also find out if two descendants hail from
the same ancestor.
 Who Invented it?
 The process of
DNA
fingerprinting was
invented by Prof.
Alec Jeffreys at
the University of
Leicester.
 Biology :
 Every cell in your body contains the same set of DNA
 DNA is unique to each individual: even though we
share 99.9% of our genome in common with other
humans, 0.1% of 3 billion nucleotides is a significant
and detectable level of difference (1 out of every 1000
nucleotides)
 Most variation exists in non-coding (viz. “junk DNA”)
regions.
 Mutations in the non-coding are tolerated and can
accumulate with no effect on the organism
 The challenge: find the differences!
 Length polymorphism

3 repeats
---------(AATG)(AATG)(AATG)----------

---------(AATG)(AATG)----------
2 repeats
 DNA Fingerprinting
 Uniquely identifies individuals on the basis of DNA fragment lengths.
– Fragments are generated by restriction enzymes that cut DNA at
specific sites.
– Each individual’s DNA is different enough that these enzymes will
generate different lengths of fragments in two different individuals.

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 Restriction Endonucleases
Also called restriction enzymes

1962: “molecular scissors” discovered in in bacteria

E. coli bacteria have an enzymatic immune system that

recognizes and destroys foreign DNA

3,000 enzymes have been identified, around 200 have

unique properties, many are purified and available
commercially
 Restriction Endonucleases

Named for bacterial genus, species, strain, and type

Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1
 Restriction Endonucleases

Recognition sites have symmetry (palindromic)

“Able was I, ere, I saw Elba”

5’-GGATCC-3’
Bam H1 site:
3’-CCTAGG-5’
5’ GGATCC 3’
CCTAGG
5’
3’

3’
5’ G GATCC
G 5’
3’ CCTAG

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 Restriction Endonucleases
Enzymes recognize specific 4-8 bp sequences

Some enzymes cut in a staggered fashion - “sticky ends”

EcoRI 5’…GAATTC…3’
3’…CTTAAG…5’

Some enzymes cut in a direct fashion – “blunt ends”

PvuII 5’…CAGCTG…3’
3’…GTCGAC…5’
 What kinds of bonds
are broken when
restriction enzymes
cut?
 Covalent bonds (within
a single strand)
 Hydrogen bonds
(between strands) as a
result of the strands
Hydrogen
coming apart bond
Covalent bond
 Variable Number Tandem
Repeats (VNTR)
 Variable number tandem
repeats are regions of DNA
that are repetitive
sequences.
 Each person has a slightly
different number of repeats.
 Therefore, if these regions
are cut with restriction
enzymes, each person will
have a different set of
fragments.

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 Which of the suspects were at the crime
scene?
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 A family consists of a mom and
dad, two daughters and two sons.
The parents have one daughter and
one son together, one daughter is
from the mother’s previous
marriage, and one son is adopted,
sharing no genetic material with
either parent. After amplifying the
VNTR DNA from each member of
the family, it is cut with a restriction
enzyme and run on an agarose gel.
The results are illustrated on the
right:

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 Other Techniques Used in DNA
Fingerprinting

 Polymerase chain reaction

– DNA replication in a test tube
– Uses primers that dictate the region of DNA to be
copied
– Makes many copies of a particular segment of
DNA
– This creates enough material for scientists to
perform DNA fingerprinting.

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 Polymerase Chain Reaction

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 Other Techniques Used in DNA
Fingerprinting

 Electrophoresis
– A way to separate DNA fragments based on their
length
– DNA sample is loaded into a gel matrix and an
electrical current is applied.
– Smaller fragments travel through the gel faster.
– Creates a banding pattern of fragments

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 Electrophoresis

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 DNA Fingerprinting Technique

 DNA is obtained from two sources:

– Blood at a crime scene
– Hair from the crime suspect
 Polymerase chain reaction is used to make many
copies of a VNTR region.
 Restriction enzymes are used to cut the VNTRs into
fragments.
 The fragments are separated by electrophoresis.
 The patterns of the two DNA samples are compared.

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 DNA Fingerprinting Applications

 DNA fingerprinting can identify if two samples

of DNA came from the same person.
 Used in the prosecution of crime suspects
 Used in paternity cases

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 DNA Fingerprinting
in a Crime Case

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 DNA Fingerprinting
in a Paternity Case

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11-27 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 DNA Sequencing

 A specially engineered DNA replication reaction.

– The DNA of interest is the template.
– Uses DNA polymerase
– Uses a primer (gives DNA polymerase a place to start)
– Uses regular nucleotides (A,T,G,C)
– Uses special fluorescently-labeled dideoxynucleotides
 When these are added to the growing chain, replication will
stop.
 The fragment will be fluorescently labeled as well.
– Fragments are separated by electrophoresis, and the
sequence can be read.
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 DNA Sequencing

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 Genetic Modification
of Organisms
 It is now possible to clone genes and move them from
one organism to another.
– Called gene cloning
 DNA sequences can be altered (mutated) to generate a
desired change.
– The new DNA is called recombinant DNA.
 Once the DNA is transferred, the new host cell begins to
make the new DNA and produce the new proteins.
– Organisms that contain recombinant DNA are called
“genetically modified organisms”.
– Usually involves bacteria or viruses that will make
large amounts of the protein of interest
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 How to Clone a Gene

 Cut the gene of interest out of the

chromosome using restriction enzymes.

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 How to Clone a Gene

 Splice the fragment containing

the gene into a carrier
molecule, usually a bacterial
plasmid.
 Plasmids ( closed circular
molecules) are DNA vehicles
which contain ori.
 Antibiotic resistant gene
 Cut the plasmid with the same
RE as you have used for GOI
 Ligase will join the gene of
interest to the plasmid

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 ori Antibiotic
resistant
gene
(Kan)

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 How to Clone a Gene

 Insert the plasmid with the fragment into the bacterial

cells.

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 How to Clone a Gene

 Each time the bacterial cells divide, many copies of

the gene will be made.

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 Human Insulin from Bacteria

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 Genetically Modified Organisms

 Genetically modified organisms have been

used to:
– Make human insulin
– Generate “Golden rice”
– Make interferon
– Make human growth hormone

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 Genetically Modified Organisms

– For bioremediation (the use of living organisms to

remove toxins from the environment)
– Generate crops that supply developing nations
with nutrients not normally found in their native
plants
– Generate crops that can manufacture medicines
to treat disease
– Generate crops that are resistant to herbicides or
that make their own insecticides

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